Treatment of immune-related and inflammatory diseases

ABSTRACT

Provided herein are methods of using compounds and compositions for modulating leukocytic activity, including activity of B cells and/or T cells monocytes, macrophages, and other lymphoid or myeloid-derived cell types, in immune-related diseases or inflammatory diseases. Pharmaceutical compositions and dosing regimens for use in the methods are also provided herein.

1. RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/281,594, filed on May 19, 2014, which is a continuation-in-partapplication of U.S. application Ser. No. 13/962,786, filed on Aug. 8,2013, which claims benefit of U.S. Provisional Patent Application Nos.61/722,718, filed on Nov. 5, 2012 and 61/681,491, filed on Aug. 9, 2012,all of which are hereby incorporated by reference in their entireties.

2. SEQUENCE LISTING

The present application is being filed with a Sequence Listing submittedas filename 12827-352-999_SeqListing.txt, of size 6,212 bytes, which wascreated on Oct. 16, 2013. The Sequence Listing is incorporated herein byreference in its entirety.

3. FIELD

Provided herein are methods of treating, preventing, and/or managingdiseases associated with leukocytic activity, including activity of Bcells and/or T cells, monocytes, macrophages, and other lymphoid ormyeloid-derived cell types e.g., immune-related diseases or inflammatorydiseases, comprising administering Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof, including(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.Pharmaceutical compositions and dosing regimens for such treatment,prevention, and/or management are also provided herein.

4. BACKGROUND

Inflammatory and immune-related diseases modulated by lymphocyticactivity, including activity of B cells and/or T cells, such as lupus,scleroderma, lupus pernio, sarcoidosis, Sjögren syndrome, ANCA-inducedvasculitis, anti-phospholipid syndrome and myasthenia gravis, continueto be important medical problems.

Lupus or lupus erythematosus is a collection of autoimmune disordersthat can cause chronic inflammation in various parts of the body,especially the skin, joints, blood, and kidneys. The body's immunesystem normally makes proteins called antibodies to protect the bodyagainst viruses, bacteria, and other foreign materials (i.e., antigens).In an autoimmune disorder such as lupus, the immune system loses itsability to tell the difference between antigens and its own cells andtissues and can make antibodies directed against its own cells andtissues to form immune complexes. These immune complexes can build up inthe tissues and cause inflammation, injury to tissues and/or pain. Thethree most common types of lupus include systemic lupus erythematosus(SLE), cutaneous lupus erythematosus (CLE) and drug-induced lupus. Moredetailed descriptions of lupus or lupus erythematosus can be found inWallace, 2000, The Lupus Book: A Guide for Patients and Their Families,Oxford University Press, Revised and Expanded Edition, which isincorporated by reference herein in its entirety.

Scleroderma is a rare disease with a stable incidence of approximately19 cases per 1 million persons. The exact cause of scleroderma isunknown. Abnormalities involve autoimmunity and alteration ofendothelial cell and fibroblast function. Systemic scleroderma usuallybegins with skin thickening, usually of the fingers, accompanied byRaynaud's phenomenon. Raynaud's disease typically precedes furthermanifestations of systemic scleroderma. Early in the disease theaffected skin may be puffy and soft. The usual location of greatest skinthickening and hardening is the face, hands and fingers. Sclerodactylyis frequently present. Tendon friction rubs are often palpable on examand can be painful. With more advanced disease, digital ulcers andauto-amputation may occur. Gastrointestinal dismotility is a feature,often manifested by heartburn, or by diarrhea with malabsorption orpseudo-obstruction. New onset hypertension or renal insufficiency aremanifestations of the associated vascular injury. Heart failure orarrhythmia are also possible due to cardiac fibrosis. (Hachulla E,Launay D, Diagnosis and classification of systemic sclerosis, Clin RevAllergy Immunol 2010; 40(2):78-83).

The major manifestations of scleroderma and in particular of systemicsclerosis are inappropriate excessive collagen synthesis and deposition,endothelial dysfunction, spasm, collapse and obliteration by fibrosis.In terms of diagnosis, an important clinical parameter is skinthickening proximal to the metacarpophalangeal joints. Raynaud'sphenomenon is a frequent, almost universal component of scleroderma. Itis diagnosed by color changes of the skin upon cold exposure. Ischemiaand skin thickening are symptoms of Raynaud's disease.

Sarcoidosis is a disease characterized by granuloma formation, enhancedby lymphocyte and macrophages, usually classified as a T-helper type 1response. Overproduction of tumor necrosis factor (TNF)-α, IL-8, andIL-18 by alveolar macrophages is thought to be a contributing factor tothe underlying lung inflammation. Lupus pernio is a chronic disfiguringskin manifestation of sarcoidosis, primarily affecting the face.Sarcoidosis and associated lupus pernio have limited treatment optionssuch as corticosteroids, which offer only modest benefit (Baughman R P,Judson M A, Teirstein A S, Moller D R, Lower E E. Thalidomide forchronic sarcoidosis. Chest. 2002 July; 122(1):227-32).

There remains a need for prophylactic or therapeutic drugs that can beused to treat or prevent immune-related and inflammatory diseases,including lupus, scleroderma, lupus pernio, sarcoidosis, Sjögrensyndrome, ANCA-induced vasculitis, anti-phospholipid syndrome andmyasthenia gravis.

5. SUMMARY

Provided herein are methods of treating, managing, ameliorating and/orpreventing diseases, disorders and/or conditions associated withimmune-related and inflammatory diseases comprising administering atherapeutically effective amount of a compound of formula I

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof.

In one embodiment, the compound is3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is a pharmaceutically acceptable salt of3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dionehydrochloride.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,having the following structure:

or a pharmaceutically acceptable salt, solvate, hydrate, or tautomerthereof.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is a pharmaceutically acceptable salt of(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dionehydrochloride.

In one embodiment, the compound is(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,having the following structure:

or a pharmaceutically acceptable salt, solvate, hydrate, or tautomerthereof.

In one embodiment, the compound is(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is a pharmaceutically acceptable salt of(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dionehydrochloride.

In certain embodiments, the disease is selected from lupus, scleroderma,Sjögren syndrome, ANCA-induced vasculitis, anti-phospholipid syndromeand myasthenia gravis.

In one embodiment, provided herein are methods of modulating, e.g.,reducing, leukocytic activity, including activity of B cells and/or Tcells, monocytes, macrophages, and other lymphoid or myeloid-derivedcell types, comprising contacting B cell and/or T cell with an effectiveamount of Compound I.

Also provided herein are pharmaceutical compositions, single unit dosageforms, and kits suitable for use in treating, preventing, amelioratingand/or managing diseases, disorders and/or conditions associatedimmune-related and inflammatory diseases, which comprise Compound I,optionally in combination with one or more other therapeutic agents.

In certain embodiments, Compound I is administered in combination withone or more therapeutic agents, i.e., pharmaceutical agents that aremodulators of leukocytic activity, including activity of B cells and/orT cells, monocytes, macrophages, and other lymphoid or myeloid-derivedcell types. The combinations encompass simultaneous as well assequential administration.

6. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon plasmablast and activated B cell differentiation.

FIG. 2 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cell viability during plasmablast differentiation.

FIG. 3 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon B and plasma cell transcription factor expression.

FIG. 4 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon IgG production in plasmablast cultures.

FIGS. 5A and 5B depict effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon B and plasma cell transcription factor expression.

FIG. 6 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon IgG production in B cell cultures on days 4, 7 and 10.

FIG. 7 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione,alone and in combination with prednisolone, on IgG production by invitro-differentiated plasma blasts/plasma cells.

FIG. 8 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon CD20/CD38 expression during B cell differentiation at day 7.

FIG. 9 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cell viability during plasmablast differentiation.

FIG. 10 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon B cell differentiation and function in SLE patient PBMC in vitro.

FIGS. 11A and 11B depict effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon IgG and IgM production, respectively, in B cell cultures on day 7.

FIGS. 12A and 12B depict effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon differentiation of CD19+ B cell into plasma blasts/plasma cells.

FIG. 13 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon large size cell population (gate p2) in normal B cell differentiationassay.

FIG. 14 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon plasma cell transcription factors in B cell differentiation culture.

FIGS. 15A-15E depict effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(20 nM) on plasma cell transcription factor expression in day 7 culturedB cells.

FIG. 16 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon transcription factors expression in CD38+ plasmablast/plasma cellsfrom differentiating SLE patient PBMC.

FIG. 17 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(represented as compound I in the figure) on CD44 MFI at day 7 B celldifferentiation culture.

FIG. 18 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon CD20^(high)/CD44^(high) cells at day 7 normal B cell differentiationassay.

FIG. 19 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(represented as compound I in the figure) on CD83+ cells and expressionat day 4 and day 7 B cell differentiation culture.

FIG. 20 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(represented as compound I in the figure) on Ig J chain expressionduring B cell differentiation culture.

FIG. 21 illustrates the effect of3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as absolute amount produced.

FIG. 22 illustrates the effect of3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as percentage of control.

FIG. 23 illustrates the effect of(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as absolute amount produced.

FIG. 24 illustrates the effect of(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as percentage of control.

FIG. 25 illustrates the effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as absolute amount produced.

FIG. 26 illustrates the effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon production of certain cytokines and chemokines in anti-CD3-stimulatedhuman T cells, expressed as percentage of control.

FIG. 27 illustrates inhibition of production of certain cytokines andchemokines in lipopolysaccharide-stimulated peripheral blood mononuclearcells by3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 28 illustrates enhancement of on production of certain cytokinesand chemokines in lipopolysaccharide-stimulated peripheral bloodmononuclear cells by3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 29 illustrates inhibition of on production of certain cytokines andchemokines in lipopolysaccharide-stimulated peripheral blood mononuclearcells by(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 30 illustrates enhancement of on production of certain cytokinesand chemokines in lipopolysaccharide-stimulated peripheral bloodmononuclear cells by(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 31 illustrates inhibition of on production of certain cytokines andchemokines in lipopolysaccharide-stimulated peripheral blood mononuclearcells by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 32 illustrates enhancement of on production of certain cytokinesand chemokines in lipopolysaccharide-stimulated peripheral bloodmononuclear cells by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 33 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as absolute amountproduced, by3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 34 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as absolute amountproduced, by(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 35 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as absolute amountproduced, by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 36 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as percentage of amountof IFN-gamma produced in the presence of 1 μm pomalidomide, by3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 37 illustrates enhancement of NK cell IFN-Gamma Production inResponse to Immobilized IgG and IL-2, expressed as percentage of amountof IFN-gamma produced in the presence of 1 μm pomalidomide, by(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 38 illustrates enhancement of NK cell IFN-Gamma Production inResponse to Immobilized IgG and IL-2, expressed as percentage of amountof IFN-gamma produced in the presence of 1 μm pomalidomide, by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 39 depicts effect of compounds provided herein on growthfactor-induced human umbilical vascular endothelial cell proliferation.

FIG. 40 depicts effect of compounds provided herein on growthfactor-induced human umbilical vascular endothelial cell tube formation.

FIG. 41 depicts effect of compounds provided herein on growthfactor-induced human umbilical vascular endothelial cell invasion.

FIG. 42 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon dermal thickness of lesional skin in the bleomycin dermal fibrosismouse model (prevention of inflammation driven fibrosis).

FIG. 43 depicts hematoxylin and eosin stained skin sectionphotomicrographs showing dermal thickness of lesional skin in thebleomycin dermal fibrosis mouse model (prevention of inflammation drivenfibrosis).

FIG. 44 depicts effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon the numbers of alpha-SMA+myofibroblasts in lesional skin in thebleomycin dermal fibrosis mouse model (prevention of inflammation drivenfibrosis).

FIG. 45 depicts effect of compounds tested on dermal thickness oflesional skin in the bleomycin dermal fibrosis mouse model (regressionof established fibrosis).

FIG. 46 depicts hematoxylin and eosin stained skin sectionphotomicrographs showing dermal thickness of lesional skin in thebleomycin dermal fibrosis mouse model (regression of establishedfibrosis).

FIG. 47 depicts reduction in the numbers of alpha-SMA+myofibroblasts inlesional skin in the bleomycin dermal fibrosis mouse model (regressionof established fibrosis).

FIG. 48A depicts reduction of dermal thickness by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionein TSK-1 mice.

FIG. 48B depicts reduction of relative hydroxyproline contents by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionein TSK-1 mice.

FIG. 49A depicts modulation of CTGF by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 49B depicts modulation of PAI-1 by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 49C depicts modulation of COL1A by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 49D depicts modulation of aSMA by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 49E depicts modulation of CCMP by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 49F depicts modulation of TGFB1 by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 50 depicts modulation of COL1, aSMA and FN by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 51 depicts modulation of MMP1 and PAI by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 52 depicts modulation of Dnmt1 by(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

FIG. 53 depicts the mRNA expression level of cereblon in normal and SScfibroblasts.

FIG. 54 depicts the mRNA expression level of cereblon in normal and SScskin tissues.

7. DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications are incorporated by reference in their entirety. In theevent that there is a plurality of definitions for a term herein, thosein this section prevail unless stated otherwise.

As used herein, and unless otherwise indicated, the terms “treat,”“treating” and “treatment” refer to alleviating or reducing the severityof a disease or a symptom associated with the disease or condition beingtreated.

As used herein, “prevent”, “prevention” and other forms of the wordinclude the inhibition of onset or progression of a disease or disorderor a symptom of the particular disease or disorder. In some embodiments,subjects with familial history of cancer are candidates for preventiveregimens. Generally, in the context of cancer, the term “preventing”refers to administration of the drug prior to the onset of signs orsymptoms of a cancer, particularly in subjects at risk of cancer.

As used herein, and unless otherwise indicated, the term “managing”encompasses preventing the recurrence of the particular disease ordisorder in a subject who had suffered from it, lengthening the time asubject who had suffered from the disease or disorder remains inremission, reducing mortality rates of the subjects, and/or maintaininga reduction in severity or avoidance of a symptom associated with thedisease or condition being managed.

As used herein, “subject” means an animal, typically a mammal, includinga human being. As used herein, “patient” means a human subject.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundrefer to an amount sufficient to provide a therapeutic benefit in thetreatment, prevention and/or management of a disease, to delay orminimize one or more symptoms associated with the disease or disorder tobe treated. The terms “therapeutically effective amount” and “effectiveamount” can encompass an amount that improves overall therapy, reducesor avoids symptoms or causes of disease or disorder or enhances thetherapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, the term“prophylactically effective amount” of a compound is an amountsufficient to prevent a disease or condition, or one or more symptomsassociated with the disease or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease. Theterm “prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” includes, but is not limited to, asalt of an acidic group that can be present in the compounds providedherein. Under certain acidic conditions, the compound can form a widevariety of salts with various inorganic and organic acids. The acidsthat can be used to prepare pharmaceutically acceptable salts of suchbasic compounds are those that form salts comprising pharmacologicallyacceptable anions including, but not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, bromide, iodide, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydroxynaphthoate, isethionate, lactate, lactobionate,malate, maleate, mandelate, methanesulfonate (mesylate), methylsulfate,muscate, napsylate, nitrate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, succinate, sulfate, tannate,tartrate, teoclate, triethiodide, and pamoate.

As used herein and unless otherwise indicated, the term “hydrate” meansa compound provided herein or a salt thereof, further including astoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces. The hydrates can be crystalline ornon-crystalline.

As used herein and unless otherwise indicated, the term “solvate” meansa solvate formed from the association of one or more solvent moleculesto compound provided herein. The term “solvate” includes hydrates (e.g.,monohydrate, dihydrate, trihydrate, tetrahydrate, and the like). Thesolvates can be crystalline or non-crystalline.

As used herein, and unless otherwise specified, the term “stereoisomer”encompasses all enantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds provided herein.

As used herein, and unless otherwise indicated, the term“stereomerically pure” or “enantiomerically pure” means that a compoundcomprises one stereoisomer and is substantially free of its counterstereoisomer or enantiomer. For example, a compound is stereomericallyor enantiomerically pure when the compound contains 80%, 90%, or 95% ormore of one stereoisomer and 20%, 10%, or 5% or less of the counterstereoisomer. In certain cases, a compound provided herein is consideredoptically active or stereomerically/enantiomerically pure (i.e.,substantially the R-form or substantially the S-form) with respect to achiral center when the compound is about 80% ee (enantiomeric excess) orgreater, preferably, equal to or greater than 90% ee with respect to aparticular chiral center, and more preferably 95% ee with respect to aparticular chiral center.

As used herein, and unless otherwise indicated, the term“stereomerically enriched” or “enantiomerically enriched” encompassesracemic mixtures as well as other mixtures of stereoisomers of compoundsprovided herein (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40,65/35 and 70/30).

The terms “co-administration” and “in combination with” include theadministration of two or more therapeutic agents (for example, CompoundI or a composition provided herein and another modulator of leukocyticactivity, including activity of B cells and/or T cells, monocytes,macrophages, and other lymphoid or myeloid-derived cell types or otheractive agent) either simultaneously, concurrently or sequentially withno specific time limits. In one embodiment, Compound I and at least oneother agent are present in the cell or in the subject's body at the sametime or exert their biological or therapeutic effect at the same time.In one embodiment, the therapeutic agent(s) are in the same compositionor unit dosage form. In another embodiment, the therapeutic agent(s) arein separate compositions or unit dosage forms.

A “B cell” is a lymphocyte that matures within the bone marrow, andincludes a naive B cell, memory B cell, or effector B cell (plasmacells). The B cell herein may be a normal or non-malignant B cell.

A “T cell” is a lymphocyte that matures in thymus, and includes a helperT cell, a memory T cell, and a cytotoxic T cell.

As used herein “overall survival” refers to the time from randomizationuntil death from any cause, and is measured in the intent-to-treatpopulation. Overall survival can be evaluated in randomized controlledstudies.

As used herein “objective response rate” refers to the proportion ofpatients with reduced predefined scleroderma symptoms at the end of apredefined period of time. Response duration is usually measured fromthe time of initial response until documented scleroderma progression.

As used herein “time to progression” means the time from randomizationuntil objective scleroderma progression. In certain embodiments, time toprogression does not include deaths.

As used herein “progression-free survival” means the time fromrandomization until objective scleroderma progression or death.

As used herein “time-to-treatment failure” means any endpoint(s)measuring time from randomization to discontinuation of treatment forany reason, including disease progression, treatment toxicity, anddeath.

As used herein “mortality” means a measure of the number of deaths in agiven population.

As used herein “respiratory mortality” means patients who die from acutehypoxemia or other specific respiratory deterioration resulting in deathsuch as need for mechanical ventilation leading to death, respiratoryarrest, or any other event in a subject deemed to be respiratory innature.

As used herein “respiratory hospitalization” means those hospitalizedfor deterioration in pulmonary status as documented by patient hospitaladmission notes or other medical opinion.

As used herein “modified Rodnan skin score” means a validated numericalscoring system to assess dermal skin thickness.

As used herein “skin thickness” means hard or indurated skin that can beevaluated using a variety of techniques including durometer and mRSS.

As used herein “skin induration” means skin that is hardened, red,inflamed, thickened or tender.

As used herein “dermatology quality of life index” means an evaluationof quality or life related to the skin symptoms for a patient havingscleroderma.

As used herein “pulmonary function” means any measurement of forcedexpiratory flow, forced vital capacity, FEV 25-75%, lung volumes orvital capacity.

As used herein “carbon monoxide diffusing capacity” means an assessmentof the uptake of carbon monoxide across the alveolar-capillary membrane.It can be a proxy for the measurement of the lungs ability to transferoxygen from the lungs to the blood stream.

As used herein “Mahler Dyspnea index” means an instrument that providesclinical measurement of shortness of breath.

As used herein “Saint George's Respiratory Questionnaire score” means aninstrument that measures quality of life in patients with pulmonarydisease.

As used herein “UCLA scleroderma clinical trial consortiumgastrointestinal tract score” means a questionnaire administered topatients having scleroderma to evaluate gastrointestinal symptomsassociated with scleroderma (systemic sclerosis).

As used herein “flow-mediated dilatation” means any measurement ofvascular endothelial function in a patient having scleroderma.

As used herein “six minute walk distance” means any evaluation of thedistance a patient having scleroderma can walk within 6 minutes or anystandardized procedure to evaluate ability to walk for a fixed period oftime or distance.

As used herein “Baseline and Transition Dyspnea Indices (BDI/TDI)” meansan interview-based measurements of breathlessness related to activitiesof daily living.

As used herein “index lesion” is a single, defined area of cutaneousinvolvement that exhibits the most severe and active sarcoidosis. Theindex lesion is located in such as position as to facilitate visualevaluation and measurement. Visual evaluation and measurement include,but are not limited to, observation by gross visual examination,microscopical observation via dermoscopy, and evauation and measurementby histology.

As used herein “Modified Sarcoidosis Activity and Severity Index” meansa validated numerical scoring system to assess sarcoidosis activity andseverity in a patient. The index includes, but is not limited to, scoresfor the symptom domains of erythema, induration and desquamation andarea scores of the index lesion.

As used herein “Physician's and Subject's Global Assessment of DiseaseActivity score” means an instrument that provides clinical measurementof sarcoidosis activity.

As used herein “6-Minute Walking Test score” means an instrument thatprovides clinical measurement of shortness of breath of a patient ofsarcoidosis after walking for 6 minutes.

As used herein “Functional Assessment of Chronic Illness Therapy-Fatigue(FACIT-F) score” means an instrument that provides clinical measurementof fatigue of a patient with sarcoidosis.

As used herein “Physician's Overall Skin Response Assessment score”means an instrument that provides clinical assessment of a target lesionin a patient with sarcoidosis.

As used herein “FVC” means the total amount of air that can be forciblyblown out after full inspiration. FVC is measured in liters.

As used herein “FEV₁” means the amount of air that can be forcibly blownout in one second. FEV₁ is measured in liters.

As used herein “chest radiographic Likert score” means an instrumentthat provides clinical assessment of the chest condition in a patientwith sarcoidosis.

As used herein, pomalidomide refers to the following compound:

7.1 Compound I

In certain embodiments, Compound I for use in the methods providedherein, including the combination therapy, and in compositions providedherein is a compound of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,having the following structure:

or a pharmaceutically acceptable salt, solvate, hydrate, or tautomerthereof.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is a pharmaceutically acceptable salt of(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dionehydrochloride.

In one embodiment, the compound is(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,having the following structure:

or a pharmaceutically acceptable salt, solvate, hydrate, or tautomerthereof.

In one embodiment, the compound is(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is a pharmaceutically acceptable salt of(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In one embodiment, the compound is selected from3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionehydrochloride,(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione,(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionehydrochloride,(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneand(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionehydrochloride.

Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof can be prepared bymethods known to one of skill in the art, for example, according to theprocedure described in US Publication No. 2011/0196150, the entirety ofwhich is incorporated herein by reference.

An exemplary method for preparation is described in Example 1.

7.2 Methods of Treatment

Provided herein are methods of treating, preventing, and/or managingdiseases, disorders and/or conditions associated with immune-related andinflammatory diseases comprising administering a therapeuticallyeffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient in need thereof. In certain embodiments, the disease isselected from lupus, scleroderma, Sjögren syndrome, ANCA-inducedvasculitis, anti-phospholipid syndrome and myasthenia gravis. In certainembodiments, the disease is lupus or scleroderma.

The sensitivity of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof canbe studied in various in vivo and in vitro assays, including animalmodels known to one of skill in the art for immune-related andinflammatory diseases, including, but not limited to MRL/MpJ-Faslpr/Jmouse model of systemic lupus erythematosus, NZBWF1/J mouse model ofsystemic lupus erythematosus, bleomycin-induced skin fibrosis model, andmurine tight skin-1 (Tsk-1) mouse model.

7.2.1 Treatment of Scleroderma

In certain embodiments, provided herein are methods of treating,preventing, and/or managing scleroderma or a symptom thereof, comprisingadministering a therapeutically effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to a patient having scleroderma. Inone embodiment, provided herein are methods of treating, preventing,and/or managing scleroderma or a symptom thereof, comprisingadministering an effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof.

In certain embodiments, provided herein are methods of preventingscleroderma or a symptom thereof, comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to a patientat risk of having scleroderma. In one embodiment, provided herein aremethods of preventing scleroderma or a symptom thereof, comprisingadministering an effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof.

In certain embodiments, the scleroderma is localized, systemic, limitedor diffuse scleroderma.

In certain embodiments, the systemic scleroderma comprises CRESTsyndrome (Calcinosis, Raynaud's syndrome, esophagaeal dysfunction ordysmotility, sclerodactyly, telangiectasia). Scleroderma is also knownas systemic sclerosis or progressive systemic sclerosis. In certainembodiments, provided herein are methods of treating or preventingRaynaud's disease or syndrome. In certain embodiments, systemicsclerosis comprises scleroderma lung disease, scleroderma renal crisis,cardiac manifestations, muscular weakness (including fatigue or limitedCREST), gastrointestinal dysmotility and spasm, and abnormalities in thecentral, peripheral and autonomic nervous system (including carpaltunnel syndrome followed by trigeminal neuralgia). It also includesgeneral disability, including depression, and impact on quality of life.

In certain embodiments, limited scleroderma is limited to the hands, theface, neck, or combinations thereof.

In certain embodiments, diffuse scleroderma comprises skin tighteningand also occurs above the wrists (or elbows). In certain embodiments,the diffuse systemic sclerosis is sine scleroderma, comprising internalorgan fibrosis, but no skin tightening; or familial progressive systemicsclerosis.

In one embodiment, scleroderma is not associated with wasting, such asdisease-related wasting.

In one embodiment, provided herein are methods for the reduction,inhibition, or prevention of one or more of the following symptoms ofscleroderma: (i) gradual hardening, thickening, and tightening of theskin (e.g., in extremities, such as hands, face, and feet); (ii) skindiscoloration; (iii) numbness of extremities; (iv) shiny skin; (v) smallwhite lumps under the surface of the skin that erupt into a chalky whitefluid; (vi) Raynaud's esophagaeal dysfunction (pain, numbness, and/orcolor changes in the hands caused by spasm of the blood vessels uponexposure to cold or emotional stress); (vii) telangiectasia (red spotson, e.g., the hands, palms, forearms, face, and lips); (viii) painand/or stiffness of the joints; (ix) swelling of the hands and feet; (x)itching of the skin; (xi) stiffening and curling of the fingers; (xii)ulcers (sores) on the outside of certain joints, such as knuckles andelbows; (xiii) digestive problems, such as heartburn, difficulty inswallowing, diarrhea, irritable bowel, and constipation; (xiv) fatigueand weakness; (xv) shortness of breath; (xvi) arthritis; (xvii) hairloss; (xviii) internal organ problems; (xix) digital ulcers; or (xx)digital auto-amputation, comprising administering an effective amount ofCompound I to a patient in need thereof.

Without being bound to any particular theory, it is believed thatCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof enhances Th1 immuneresponse, and suppresses Th2 immune response, which may result inanti-fibrotic effects in the skin.

Further provided herein are methods for improving or reducing the skinthickness of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the skin thickness is reduced by about20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%about 80%, about 90% or more.

Further provided herein are methods for achieving one or more clinicalendpoints associated with scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient in need thereof.

Further provided herein are methods for increasing the overall survival,objective response rate, time to progression, progression-free survivaland/or time-to-treatment failure of a patient having sclerodermacomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for decreasing mortality,respiratory mortality and/or respiratory hospitalization of a patienthaving scleroderma comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for improving the modified Rodnanskin score of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the improvement in modified Rodnan skinscore is 5, 10, 15 or 20 points or more.

Further provided herein are methods for improving or reducing the skinthickness of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the skin thickness is reduced by about20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%about 80%, about 90% or more.

Further provided herein are methods for improving or reducing skininduration of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient.

Further provided herein are methods for improving the dermatologyquality of life index of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

Further provided herein are methods for improving the pulmonary functionof a patient having scleroderma comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to thepatient.

Further provided herein are methods for improving the carbon monoxidediffusing capacity of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the carbon monoxidediffusing capacity of a patient is improved by an improvement in thediffusing capacity of the lung for carbon monoxide (D_(L)co) of about10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%,about 70% about 80%, about 90% or more.

Further provided herein are methods for improving the Mahler Dyspneaindex of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the improvement in Mahler Dyspnea indexis 4, 5, 6, 7, 8, 9 or 10 points or more.

Further provided herein are methods for improving the Saint George'sRespiratory Questionnaire score of a patient having sclerodermacomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the improvement in Saint George's Respiratory Questionnaire score is 4,8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 points or more.

Further provided herein are methods for improving the UCLA sclerodermaclinical trial consortium gastrointestinal tract score of a patienthaving scleroderma comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for treating or preventing digitalulcer of a patient or patient population having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

Further provided herein are methods improving flow-mediated dilatationof a patient having scleroderma comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to thepatient.

Further provided herein are methods improving or increasing the sixminute walk distance of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the improvement inthe six minute walk distance is about 200 meters, about 250 meters,about 300 meters, about 350 meters, about 400 meters or more.

7.2.2 Treatment of Lupus Erythematosus

In certain embodiments, provided herein are methods of treating,preventing, and/or managing lupus erythematosus or a symptom thereof,comprising administering a therapeutically effective amount of CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to a patient having lupuserythematosus. In one embodiment, provided herein are methods oftreating, preventing, and/or managing lupus erythematosus or a symptomthereof, comprising administering a therapeutically effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof to a patient having lupuserythematosus.

In one embodiment, provided herein are methods of preventing lupuserythematosus or a symptom thereof, comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient at risk of having lupus erythematosus. In one embodiment,provided herein are methods of preventing lupus erythematosus or asymptom thereof, comprising administering an effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof to a patient at risk ofhaving lupus erythematosus.

In certain embodiments, provided herein are methods for treating,preventing, and/or managing systemic lupus erythematosus (SLE),cutaneous lupus erythematosus (CLE) or drug-induced lupus.

The phrase “Systemic lupus erythematosus” is interchangeably used hereinwith SLE and lupus and refers to all manifestations of the disease asknown in the art (including remissions and flares). In SLE, abnormalhyperactivity of B lymphocytes and massive abnormal production ofimmunoglobulin gamma (IgG) auto-antibodies play a key role. Thispathological process results in sequestration and destruction ofIg-coated cells, fixation and cleaving of complement proteins, andrelease of chemotaxins, vasoactive peptides and destructive enzymes intotissues (Hahn B H. Systemic Lupus Erythematosus. In: Kasper D L,Braunwald E, Fauci A S, Hauser S L, Longo D L, Jameson, J L, editors.In: Harrison's Principles of Internal Medicine (16th edition). New York(US): McGraw-Hill; 2005. pp. 1960-1967).

Symptoms of SLE vary from person to person, and may come and go. In mostpatients, the symptoms include joint pain and swelling. Frequentlyaffected joints are the fingers, hands, wrists, and knees. Some patientsdevelop arthritis. Other common symptoms include: chest pain when takinga deep breath, fatigue, fever with no other cause, general discomfort,uneasiness, or ill feeling (malaise), hair loss, mouth sores, swollenlymph nodes, sensitivity to sunlight, skin rash-a “butterfly” rash overthe cheeks and bridge of the nose affects about half of people with SLE,in some patients, the rash gets worse in sunlight, and the rash may alsobe widespread.

Other symptoms depend on what part of the body is affected, and mayinclude the following:

-   -   Brain and nervous system: headaches, numbness, tingling,        seizures, vision problems, personality changes,    -   Digestive tract: abdominal pain, nausea, and vomiting,    -   Heart: abnormal heart rhythms (arrhythmias),    -   Lung: coughing up blood and difficulty breathing, and    -   Skin: patchy skin color, fingers that change color when cold        (Raynaud's phenomenon).

Some patients only have skin symptoms. This is called discoid lupus.

In one embodiment, provided herein are methods of treating moderate,severe, or very severe SLE. The term “severe SLE” as used herein refersto an SLE condition where the patient has one or more severe orlife-threatening symptoms (such as hemolytic anemia, extensive heart orlung involvement, kidney disease, or central nervous systeminvolvement).

Further provided herein are methods for achieving one or more clinicalendpoints associated with SLE comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to a patientin need thereof.

Further provided herein are methods for increasing the overall survival,objective response rate, time to progression, progression-free survivaland/or time-to-treatment failure of a patient having SLE comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

In certain embodiment, Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereofacts as an inhibitor of primary human memory CD19+ B-celldifferentiation to the plasmablast stage. Without being bound to anyparticular theory, it is believed that Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof blocks cells at a premature stage thereby decreasingthe numbers of plasmablasts that are capable of producing high levels ofimmunoglobulin. A functional consequence of this effect is reducedimmunoglobulin G (IgG) and immunoglobulin M (IgM) production in thesedifferentiation cultures.

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof inhibits of the ability of primary human memory CD19+ B-cells todifferentiate to the plasmablast stage. In certain embodiments, CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof has no significant effect on matureCD138+ plasma cells in short term cultures. In certain embodiments,Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof inhibits B celldifferentiation factors including interferon regulatory factor 4 (TRF4),lymphocyte-induced maturation protein (BLIMP), X-box-protein-1 (XBP-1)and B cell lymphoma 6 (Bcl6).

7.2.3 Treatment of Sarcoidosis

In certain embodiments, provided herein are methods of treating,preventing, and/or managing sarcoidosis or a symptom thereof, comprisingadministering a therapeutically effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to a patient having sarcoidosis. Inone embodiment, provided herein are methods of treating, preventing,and/or managing sarcoidosis or a symptom thereof, comprisingadministering an effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof.

In certain embodiments, provided herein are methods of preventingsarcoidosis or a symptom thereof, comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to a patientat risk of having sarcoidosis. In one embodiment, provided herein aremethods of preventing sarcoidosis or a symptom thereof, comprisingadministering an effective amount of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof.

Sarcoidosis is a disease characterized by granuloma formation, enhancedby lymphocyte and macrophages, usually classified as a T-helper type 1response. Sarcoidosis patients often develop damage in a number oforgans including, but not limited to, pulmonary, CNS, GI, lymphatics,optic, and renal systems. In one embodiment, the sarcoidosis is withlung involvement (e.g., pulmonary sarcoid), with skin involvement (e.g.,cutaneous sarcoid), with heart involvement, or with brain involvement.In certain embodiments, the sarcoidosis is chronic cutaneous sarcoid(CCS). In one embodiment, the CCS is with pulmonary involvement. Inanother embodiment, the CCS is without pulmonary involvement. As usedherein, sarcoidosis also includes the diseases associated withsarcoidosis (e.g., lupus pernio).

In one embodiment, provided herein are methods for the reduction,inhibition, or prevention of one or more of the following symptoms ofsarcoidosis: (i) granulomas formulation; (ii) lupus pernio; (iii) skinlesions; (iv) dyspnea; (v) dry cough; (vi) chest pain, and (vii)fatigue, comprising administering an effective amount of Compound I to apatient in need thereof.

In certain embodiments, the patient has been diagnosed with chroniccutaneous sarcoid for at least 12 months. In some embodiments, thediagnosis of chronic cutaneous sarcoid is confirmed by cutaneous sarcoidbiopsy. In certain embodiments, the patient has a Minimum ModifiedSarcoidosis Activity and Severity Index (MSASI) of ≥1. In certainembodiments, the patient has at least 2 lesions >1 cm in longestdimension. In certain embodiments, the patient has no improvement fromanother chronic cutaneous sarcoid treatment for at least 3 months. Incertain embodiments, the patient has a forced vital capacity (FVC) of≥45% of predicted normal value. In certain embodiments, the patient hasan estimated Glomerular Filtration Rate (eGFR) ≥60 mL/min. In someembodiments, the eGFR is calculated by MDRD [Modification of Diet inRenal Disease].

Further provided herein are methods for achieving one or more clinicalendpoints associated with sarcoidosis comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient in need thereof.

Further provided herein are methods for increasing the overall survival,objective response rate, time to progression, progression-free survivaland/or time-to-treatment failure of a patient having sarcoidosiscomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient.

In certain embodiments, the clinical endpoint is improvement in theModified Sarcoidosis Activity and Severity Index (MSASI) score of thepatient having sarcoidosis. In some embodiments, provided herein aremethods for improving or increasing the Modified Sarcoidosis Activityand Severity Index (MSASI) score of a patient having sarcoidosiscomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the symptom domains of erythema, induration and desquamation are scoredas follows: 0=none; 1=slight; 2=moderate; 3=severe; and 4=very severe.In one embodiment, the symptom score for domains of erythema, indurationand/or desquamation in the patient is improved by at least 1, at least2, at least 3, or at least 4 levels. In one embodiment, the area scoreof the index lesion is assessed as follows: 0=0%; 1=1-9%; 2=10-29%;3=30-49%; 4=50-69%; 5=70-89%; and 6=90-100%. In one embodiment, the areascore of the index lesion is improved by at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or more.

In certain embodiments, the clinical endpoint is improvement in thePhysician's and Subject's Global Assessment of Disease Activity score ofthe patient having sarcoidosis. In some embodiments, provided herein aremethods for improving or increasing the Physician's and Subject's GlobalAssessment of Disease Activity score of a patient having sarcoidosiscomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the Physician's and Subject's Global Assessment of Disease Activityscore of the patient is improved by at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or more.

In certain embodiments, the clinical endpoint is improvement in the6-Minute Walking Test (6WMT) score of the patient having sarcoidosis. Insome embodiments, provided herein are methods for improving orincreasing the 6WMT score of a patient having sarcoidosis comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the 6WMT score isimproved by at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, or at least 9 levels based on theBorg Scale.

In certain embodiments, the clinical endpoint is improvement in theFunctional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) scoreof the patient having sarcoidosis. In some embodiments, provided hereinare methods for improving or increasing the FACIT-F score of a patienthaving sarcoidosis comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient. Inone embodiment, the FACIT-F score of the patient is improved by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or more.

In certain embodiments, the clinical endpoint is improvement in theDermoscopy for Lesion Assessment score of the patient havingsarcoidosis. In some embodiments, provided herein are methods forimproving or increasing the Dermoscopy for Lesion Assessment score of apatient having sarcoidosis comprising administering an effective amountof Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient. Inone embodiment, the Dermoscopy for Lesion Assessment score of thepatient is improved by at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, ormore.

In certain embodiments, the clinical endpoint is improvement in thePhysician's Overall Skin Response Assessment score of the patient havingsarcoidosis. In some embodiments, provided herein are methods forimproving or increasing the Physician's Overall Skin Response Assessmentscore of a patient having sarcoidosis comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the Physician's Overall Skin ResponseAssessment is scored as follows: 1=markedly worsened; 2=worsened;3=unchanged; 4=near resolution; and 5=complete resolution. In oneembodiment, the Physician's Overall Skin Response Assessment score inthe patient is improved by at least 1, at least 2, at least 3, or atleast 4 levels.

In certain embodiments, the clinical endpoint is improvement in lesionof the patient having sarcoidosis as determined by photographicassessment. In some embodiments, provided herein are methods forimproving lesion of a patient having sarcoidosis as determined byphotographic assessment comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient. Inone embodiment, the lesion of the patient is improved by at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or more.

In certain embodiments, the clinical endpoint is improvement in lesionof the patient having sarcoidosis as determined by skin biopsy. In someembodiments, provided herein are methods for improving lesion of apatient having sarcoidosis as determined by skin biopsy comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the lesion is anon-facial lesion. In one embodiment, the improvement of lesion ischaracterized as one or more of changes in granuloma size and number,and epidermal thickness, and IHC staining for cells such as Giant cells,T cell staining (e.g., CD4 and CD8), and Dendritic cells/Langerhansstaining (e.g., CD1a, CD83, DC-LAMP). In one embodiment, the lesion ofthe patient is improved by at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, ormore.

In certain embodiments, the clinical endpoint is improvement in one ormore biomarkers associated with sarcoidosis of the patient havingsarcoidosis. In some embodiments, provided herein are methods forimproving, increasing, or decreasing one or more biomarkers associatedwith sarcoidosis of a patient having sarcoidosis comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the biomarker is aserum biomarker. In one embodiment, the biomarker is serum ACE, IgG,25-hydroxy vitamin D (25-OH-vit D), or 1,25-dihydroxy vitamin D(1,25-vitD). In one embodiment, the biomarker is a plasma biomarker. Inone embodiment, the biomarker is IL-12, sIL2R, or TNF-α. In oneembodiment, the biomarker is peripheral blood total lymphocyte countsand B cell subsets and T cell activation markers (such as but notlimited to HLA-DR, CD45RA, CD45RO, CD69, IL-2R, CD80, CD86).

In certain embodiments, the clinical endpoint is improvement in FVC orFEV₁ of the patient having sarcoidosis. In some embodiments, providedherein are methods for improving or increasing FVC or FEV₁ of a patienthaving sarcoidosis comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient. Inone embodiment, the FVC or FEV₁ of the patient is improved by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or more.

In certain embodiments, the clinical endpoint is improvement in thechest radiographic Likert score of the patient having sarcoidosis. Insome embodiments, provided herein are methods for improving orincreasing the chest radiographic Likert score of a patient havingsarcoidosis comprising administering an effective amount of Compound Ior a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the chest radiographic Likert score is on a Likert scale of 1 to 5(markedly worsened, worsened, unchanged, improved, and markedlyimproved). In one embodiment, the chest radiographic Likert score in thepatient is improved by at least 1, at least 2, at least 3, or at least 4levels.

In certain embodiments, the clinical endpoint is improvement in theSaint George's Respiratory Questionnaire score in the patient havingsarcoidosis. Further provided herein are methods for improving the SaintGeorge's Respiratory Questionnaire score of a patient having sarcoidosiscomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the improvement in Saint George's Respiratory Questionnaire score is 4,8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 points or more.

In certain embodiments, the clinical endpoint is improvement in thedyspnea index (BDI)/transition dyspnea index (TDI) in the patient havingsarcoidosis. In some embodiments, provided herein are methods forimproving or increasing the dyspnea index (BDI)/transition dyspnea index(TDI) of a patient having sarcoidosis comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, at baseline, dyspnea is rated using fivegrades from 0 (severe) to 4 (unimpaired) for each of three categories(baseline functional impairment, baseline magnitude of task, andbaseline magnitude of effort) to form a baseline score (0 to 12); duringthe transition period, the TDI assesses changes in dyspnea using sevengrades ranging from −3 (major deterioration) to +3 (major improvement);and the ratings for each of the three categories are then added to forma total transition focal score (range, −9 to +9). In one embodiment, thedyspnea index (BDI)/transition dyspnea index (TDI) in the patient isimproved by at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, or 9 levels.

In one embodiment, the improvement provided herein is measured after thetreatment for a period of time. In one embodiment, the treatment time is1 week, 2 weeks, 4 weeks, 6 weeks, 12 weeks, 6 months, or a year. In oneembodiment, the treatment time is 12 weeks.

In certain embodiments, the dosage of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof is in the range of from about 0.1 mg QD to about 1 mgQD. In one embodiment, the dosage is in the range of from about 0.3 mgQD to about 0.6 mg QD. In one embodiment, the dosage is about 0.3 mg QD.In another embodiment, the dosage is about 0.6 mg QD.

7.2.4 Treatment of Other Immune-Related Diseases or Disorders

Further provided herein are methods of treating, managing, or preventingother immune-related diseases or conditions using Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof. In certain embodiments, providedherein are methods of treating, managing, or preventing otherimmune-related diseases or conditions using(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof. In certain embodiments,for example, provided herein is a method of treating an individualhaving a disease or disorder, wherein the disease or disorder is causedby, or is associated with, an inappropriate or undesirable immuneresponse, e.g., a disease, disorder or condition that can be treatedbeneficially by immunosuppression, comprising administering to theindividual Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof. In certainembodiments, provided herein is a method of treating an individualhaving a disease or disorder, wherein the disease or disorder is causedby, or is associated with, an inappropriate or undesirable immuneresponse, e.g., a disease, disorder or condition that can be treatedbeneficially by immunosuppression, comprising administering to theindividual(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof.

In various specific embodiments, said immune-related disease is one ormore of selected from Sjögren syndrome, ANCA-induced vasculitis,anti-phospholipid syndrome, myasthenia gravis, Addison's disease,alopecia areata, ankylosing spondylitis, antiphospholipid antibodysyndrome, antiphospholipid syndrome (primary or secondary), asthma,autoimmune gastritis, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease, autoimmune lymphoproliferative disease,autoimmune thrombocytopenic purpura, Balo disease, Behcet's disease,bullous pemphigoid, cardiomyopathy, celiac disease, Chagas disease,chronic inflammatory demyelinating polyneuropathy, cicatrical pemphigoid(e.g., mucous membrane pemphigoid), cold agglutinin disease, degosdisease, dermatitis hepatiformis, essential mixed cryoglobulinemia,Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,Hashimoto's thyroiditis (Hashimoto's disease; autoimmune thyroditis),idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgAnephropathy, juvenile arthritis, lichen planus, Meniere disease, mixedconnective tissue disease, morephea, narcolepsy, neuromyotonia,pediatric autoimmune neuropsychiatric disorders (PANDAs), pemphigusvulgaris, pernicious anemia, polyarteritis nodosa, polychondritis,polymyalgia rheumatica, primary agammaglobulinemia, primary biliarycirrhosis, Raynaud disease (Raynaud phenomenon), Reiter's syndrome,relapsing polychondritis, rheumatic fever, Sjogren's syndrome,stiff-person syndrome (Moersch-Woltmann syndrome), Takayasu's arteritis,temporal arteritis (giant cell arteritis), uveitis, vasculitis (e.g.,vasculitis not associated with lupus erythematosus), vitiligo, and/orWegener's granulomatosis.

7.2.5 Treatment for Patients with Renal Impairment

In certain embodiments, provided herein are methods of treating,preventing, and/or managing a disease provided herein in patients withimpaired renal function. In certain embodiments, provided herein aremethods of providing appropriate dose adjustments for patients withimpaired renal function due to, but not limited to, disease, aging, orother patient factors.

In certain embodiments, provided herein are methods of treating,preventing, and/or managing a disease provided herein, or a symptomthereof, in patients with impaired renal function comprisingadministering a therapeutically effective amount of a compound providedherein to the patient with impaired renal function. In one embodiment,provided herein are methods of treating, preventing, and/or managingrelapsed disease, or a symptom thereof, in patients with impaired renalfunction comprising administering a therapeutically effective amount of(S)-3-(4-((4-morphlinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof to a patient havingrelapsed disease with impaired renal function.

In one embodiment, provided herein are methods of preventing a relapsein patients with impaired renal function, comprising administering aneffective amount of a compound provided herein to a patient withimpaired renal function at risk of having a relapse. In one embodiment,provided herein are methods of preventing a relapse in patients withimpaired renal function, comprising administering an effective amount of(S)-3-(4-((4-morphlinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof, to a patient withimpaired renal function at risk of having a relapse.

In all of the embodiments provided herein, when a renally impairedpatient is treated, there is a need for administering to the renallyimpaired patient a dose of the compound lower than the dose administeredto a normal patient (e.g., a patient without renal impairment) becauseof the decreased ability of the renally impaired patient in eliminatingpomalidomide or its metabolites. Thus, in one embodiment, providedherein is a method for treating a renally impaired patient with a doseof a compound provided herein lower than the dose administered to anormal patient.

In certain embodiments, a therapeutically or prophylactically effectiveamount of the compound is from about 0.005 to about 1,000 mg per day,from about 0.01 to about 500 mg per day, from about 0.01 to about 250 mgper day, from about 0.01 to about 100 mg per day, from about 0.1 toabout 100 mg per day, from about 0.5 to about 100 mg per day, from about1 to about 100 mg per day, from about 0.01 to about 50 mg per day, fromabout 0.1 to about 50 mg per day, from about 0.5 to about 50 mg per day,from about 1 to about 50 mg per day, from about 0.02 to about 25 mg perday, or from about 0.05 to about 10 mg per day.

7.3 Dosages and Dosing Amounts

The dose of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to beadministered to a patient is rather widely variable and can be subjectto the judgment of a health-care practitioner. Doses of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof vary depending on factors such as:specific indication to be treated, prevented, or managed; age andcondition of a patient; and amount of second active agent used, if any.In general, Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof can beadministered one to four or more times a day in a dose of about 0.005mg/kg of a patient's body weight to about 10 mg/kg of a patient's bodyweight in a patient, but the above dosage may be properly varieddepending on the age, body weight and medical condition of the patientand the type of administration. In one embodiment, the dose is about0.01 mg/kg of a patient's body weight to about 5 mg/kg of a patient'sbody weight, about 0.05 mg/kg of a patient's body weight to about 1mg/kg of a patient's body weight, about 0.1 mg/kg of a patient's bodyweight to about 0.75 mg/kg of a patient's body weight or about 0.25mg/kg of a patient's body weight to about 0.5 mg/kg of a patient's bodyweight.

In one embodiment, one dose is given per day. In any given case, theamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof administeredwill depend on such factors as the solubility of the active component,the formulation used and the route of administration. In one embodiment,application of a topical concentration provides intracellular exposuresor concentrations of about 0.01-10 μM.

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof is used in an amount of from about 0.1 mg to about 1000 mg perday, and can be adjusted in a conventional fashion (e.g., the sameamount administered each day of the treatment, prevention or managementperiod), in cycles (e.g., one week on, one week off), or in an amountthat increases or decreases over the course of treatment, prevention, ormanagement. In other embodiments, the dose can be from about 1 mg toabout 300 mg, from about 0.1 mg to about 150 mg, from about 1 mg toabout 200 mg, from about 10 mg to about 100 mg, from about 0.1 mg toabout 50 mg, from about 1 mg to about 50 mg, from about 10 mg to about50 mg, from about 20 mg to about 30 mg, or from about 1 mg to about 20mg. In other embodiments, the dose can be from about 0.1 mg to about 100mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 25 mg,from about 0.1 mg to about 20 mg, from about 0.1 mg to about 15 mg, fromabout 0.1 mg to about 10 mg, from about 0.1 mg to about 7.5 mg, fromabout 0.1 mg to about 5 mg, from about 0.1 mg to about 4 mg, from about0.1 mg to about 3 mg, from about 0.1 mg to about 2 mg, or from about 1mg to about 1 mg.

7.4 Combination Therapy

Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof can be combined withother pharmacologically active compounds (“second active agents”) inmethods and compositions provided herein. Certain combinations may worksynergistically in the treatment of particular types diseases ordisorders, and conditions and symptoms associated with such diseases ordisorders. Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof can alsowork to alleviate adverse effects associated with certain second activeagents, and vice versa.

One or more second active ingredients or agents can be used in themethods and compositions provided herein. Second active agents can belarge molecules (e.g., proteins) or small molecules (e.g., syntheticinorganic, organometallic, or organic molecules).

In another embodiment, the method of treatment provided herein comprisesthe administration of a second therapeutic agent, wherein the secondtherapeutic agent is an anti-inflammatory drug, e.g., a steroidalanti-inflammatory drug, or a non-steroidal anti-inflammatory drug(NSAID), acetaminophen, naproxen, ibuprofen, acetylsalicylic acid, andthe like. In a more specific embodiment in which an NSAID isadministered, a proton pump inhibitor (PPI), e.g., omeprazole may alsoadministered. In one embodiment, the antiinflammatory agent is acorticosteroid. In another embodiment, the antiinflammatory agent iscolchicine.

In another embodiment, the second therapeutic agent is animmunomodulatory compound or an immunosuppressant compound such asazathioprine (Imuran™, Azasan™), methotrexate (Rheumatrex™, Trexall™),penicillamine (Depen™, Cuprimine™), cyclophosphamide (Cytoxan™),mycophenalate (CellCept™, Myfortic™), bosentan (Tracleer®), prednisone(Deltasone™, Liquid Pred™), and a PDE5 inhibitor. In another embodiment,where the affected individual has digital ulcerations and pulmonaryhypertension, a vasodilator such as prostacyclin (iloprost) may beadministered.

In another embodiment, the second therapeutic agent is an HDACinhibitor, such as romidepsin, vorinostat, panobinostat, valproic acid,or belinostat; or a biological agent, such as an interleukin, animmunomodulatory monoclonal antibody, or bacillus Calmette-Guerin (BCG).

In another embodiment, the second therapeutic agent is an inhibitor ofActRII receptors or an activin-ActRII inhibitor. Inhibitors of ActRIIreceptors include ActRIIA inhibitors and ActRIIB inhibitors. Inhibitorsof ActRII receptors can be polypeptides comprising activin-bindingdomains of ActRII. In certain embodiments, the activin-binding domaincomprising polypeptides are linked to an Fc portion of an antibody(i.e., a conjugate comprising an activin-binding domain comprisingpolypeptide of an ActRII receptor and an Fc portion of an antibody isgenerated). In certain embodiments, the activin-binding domain is linkedto an Fc portion of an antibody via a linker, e.g., a peptide linker.

An exemplary activin-binding ActRIIA polypeptide fused to a human Fedomain is provided in SEQ ID NO: 1.

SEQ ID NO: 1 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An exemplary fusion protein comprising a soluble extracellular domain ofActRIIB fused to an Fe domain is provided in SEQ ID NO: 2.

SEQ ID NO: 2 ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Further examples of non-antibody proteins selected for activin orActRIIA binding and methods for design and selection of the same arefound in WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989,US 2003/0133939, and US 2005/0238646, each of which is incorporatedherein by reference in its entirety.

In one embodiment, the inhibitor of ActRII receptors is ACE-11. Inanother embodiment, the inhibitor of ActRII receptors is ACE-536.

Any combination of the above therapeutic agents, suitable for treatmentof the diseases or symptoms thereof, can be administered. Suchtherapeutic agents can be administered in any combination with CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof, at the same time or as a separatecourse of treatment.

7.5 Cycling Therapy

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof provided herein is cyclically administered to a patient. Cyclingtherapy involves the administration of an active agent for a period oftime, followed by a rest (i.e., discontinuation of the administration)for a period of time, and repeating this sequential administration.Cycling therapy can reduce the development of resistance to one or moreof the therapies, avoid or reduce the side effects of one of thetherapies, and/or improve the efficacy of the treatment.

Consequently, in one embodiment, a compound provided herein isadministered daily in a single or divided doses in a four to six weekcycle with a rest period of about a week or two weeks. Cycling therapyfurther allows the frequency, number, and length of dosing cycles to beincreased. Thus, another embodiment encompasses the administration of acompound provided herein for more cycles than are typical when it isadministered alone. In yet another embodiment, a compound providedherein is administered for a greater number of cycles than wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, a compound provided herein is administered daily andcontinuously for three or four weeks at a dose of from about 0.03 mg toabout 10 mg per day, followed by a rest of one or two weeks. In otherembodiments, the dose can be from about 0.1 mg to about 8 mg, from about0.3 mg to about 6 mg, from about 1 mg to about 4 mg, or about 2 mg,followed by a rest.

In one embodiment, a compound provided herein and a second activeingredient are administered orally, with administration of the compoundprovided herein occurring 30 to 60 minutes prior to the second activeingredient, during a cycle of four to six weeks. In another embodiment,the combination of a compound provided herein and a second activeingredient is administered by intravenous infusion over about 90 minutesevery cycle.

Typically, the number of cycles during which the combination treatmentis administered to a patient will be from about one to about 24 cycles,from about two to about 16 cycles, or from about four to about threecycles.

7.6 Biomarkers

In certain embodiments, provided herein are biomarkers for the treatmentof various disease or disorders provided herein. In one embodiment, thebiomarker is cluster of differentiation-44 (“CD44”), a molecule found onthe surface of B cells. In another embodiment, the biomarker is clusterof differentiation-83 (“CD83”), a molecule found on the surface of Bcells. In other embodiments, the biomarker is a combination of CD44 andCD83.

The levels of the protein biomarkers provided herein can be detected orquantitated by any methods known in the art. In certain embodiments,antibody-based methods are used. In certain embodiments, the detectingor quantitating method is immunoblotting (western blot), anenzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flowcytometry, a cytometric bead array, or mass spectroscopy.

In certain embodiments, the detecting or quantitating method isimmunoblotting (western blot). In certain embodiments, the detecting orquantitating method is an enzyme-linked immunosorbent assay (ELISA). Incertain embodiments, the detecting or quantitating method is a directELISA. In certain embodiments, the detecting or quantitating method isan indirect ELISA. In certain embodiments, the detecting or quantitatingmethod is an sandwich ELISA. In certain embodiments, the detecting orquantitating method is immunohistochemistry. In certain embodiments, thedetecting or quantitating method is flow cytometry. In certainembodiments, the detecting or quantitating method is a cytometric beadarray. In certain embodiments, the detecting or quantitating method ismass spectroscopy.

Certain embodiments include methods of identifying a subject who islikely to be responsive to a treatment of a disease, disorder, orcondition with a treatment compound. In certain embodiments the methodinvolves determining the level of a biomarker in a biological samplefrom the subject. In certain embodiments the biomarker is CD44. Incertain embodiments the biomarker is CD83.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44,CD83 or combination thereof, and b) comparing the level of the biomarkerin the biological sample from the subject to a reference level of thebiomarker; wherein the subject is likely to be responsive to thetreatment if the level of the biomarker in the biological sample fromthe subject is altered as compared to the reference level of thebiomarker.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44,CD83 or combination thereof, b) determining the level of the biomarkerin a control sample; and c) comparing the level of the biomarker in thebiological sample from the subject to the level of the biomarker in thecontrol sample; wherein the subject is likely to be responsive to thetreatment if the level of the biomarker in the biological sample fromthe subject is altered as compared to the level of the biomarker in thecontrol sample.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD83; andb) comparing the level of the biomarker in the biological sample fromthe subject to a reference level of the biomarker; wherein the subjectis likely to be responsive to the treatment if the level of thebiomarker in the biological sample from the subject is higher than thereference level of the biomarker.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD83; b)determining the level of the biomarker in a control sample; and c)comparing the level of the biomarker in the biological sample from thesubject to the level of the biomarker in the control sample; wherein thesubject is likely to be responsive to the treatment if the level of thebiomarker in the biological sample from the subject is higher than thelevel of the biomarker in the control sample.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44; andb) comparing the level of the biomarker in the biological sample fromthe subject to a reference level of the biomarker; wherein the subjectis likely to be responsive to the treatment if the level of thebiomarker in the biological sample from the subject is lower than thereference level of the biomarker.

In certain embodiments methods of identifying a subject who is likely tobe responsive to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44; b)determining the level of the biomarker in a control sample; and c)comparing the level of the biomarker in the biological sample from thesubject to the level of the biomarker in the control sample; wherein thesubject is likely to be responsive to the treatment if the level of thebiomarker in the biological sample from the subject is lower than thelevel of the biomarker in the control sample.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44,CD83 or combination thereof, and b) comparing the level of the biomarkerin the biological sample to a reference level of the biomarker; whereinthe difference between the level of the biomarker in the biologicalsample from the subject and the reference level of the biomarkercorrelates with the responsiveness of the subject to the treatment.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44,CD83 or combination thereof, b) determining the level of the biomarkerin a control sample; and c) comparing the level of the biomarker in thebiological sample from the subject to the level of the biomarker in thecontrol sample; wherein the difference between the level of thebiomarker in the biological sample from the subject and the level of thebiomarker in the control sample correlates with the responsiveness ofthe subject to the treatment.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD83; andb) comparing the level of the biomarker in the biological sample to areference level of the biomarker, wherein an increased level of thebiomarker in the biological sample from the subject in comparison withthe reference level of the biomarker correlates with an increasedresponsiveness of the subject to the treatment.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD83; b)determining the level of the biomarker in a control sample; and c)comparing the level of the biomarker in the biological sample from thesubject to the level of the biomarker in the control sample; wherein anincreased level of the biomarker in the biological sample from thesubject in comparison with the level of the biomarker in the controlsample correlates with an increased responsiveness of the subject to thetreatment.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44; andb) comparing the level of the biomarker in the biological sample to areference level of the biomarker, wherein a decreased level of thebiomarker in the biological sample from the subject in comparison withthe reference level of the biomarker correlates with an increasedresponsiveness of the subject to the treatment.

In certain embodiments methods of predicting the responsiveness of asubject to a treatment of a disease, disorder, or condition with atreatment compound, comprise: a) determining the level of a biomarker ina biological sample from the subject, wherein the biomarker is CD44; b)determining the level of the biomarker in a control sample; and c)comparing the level of the biomarker in the biological sample from thesubject to the level of the biomarker in the control sample; wherein adecreased level of the biomarker in the biological sample from thesubject in comparison with the level of the biomarker in the controlsample correlates with an increased responsiveness of the subject to thetreatment.

In certain embodiments methods of monitoring the efficacy of a treatmentof a disease, disorder, or condition in a subject treated with atreatment compound, comprise: a) obtaining a first biological samplefrom the subject; b) determining the level of a biomarker in the firstbiological sample, wherein the biomarker is CD 44, CD83 or combinationthereof; c) administering the treatment compound to the subject; d)thereafter obtaining a second biological sample from the subject; e)determining the level of the biomarker in the second biological sample;and f) comparing the levels of the biomarker in the first and secondbiological samples; wherein the subject is responsive to the treatmentif the level of the biomarker in the second biological sample of thesubject is altered as compared to the level of the biomarker in thefirst biological sample of the subject.

In certain embodiments methods of monitoring the efficacy of a treatmentof a disease, disorder, or condition in a subject treated with atreatment compound, comprise: a) obtaining a first biological samplefrom the subject; b) determining the level of a biomarker in the firstbiological sample, wherein the biomarker is CD83; c) administering thetreatment compound to the subject; d) thereafter obtaining a secondbiological sample from the subject; e) determining the level of thebiomarker in the second biological sample, and f) comparing the levelsof the biomarker in the first and second biological samples; wherein thesubject is responsive to the treatment if the level of the biomarker inthe second biological sample of the subject is higher than the level ofthe biomarker in the first biological sample of the subject.

In certain embodiments methods of monitoring the efficacy of a treatmentof a disease, disorder, or condition in a subject treated with atreatment compound, comprise: a) obtaining a first biological samplefrom the subject; b) determining the level of a biomarker in the firstbiological sample, wherein the biomarker is CD44; c) administering thetreatment compound to the subject; d) thereafter obtaining a secondbiological sample from the subject; e) determining the level of thebiomarker in the second biological sample, and f) comparing the levelsof the biomarker in the first and second biological samples; wherein thesubject is responsive to the treatment if the level of the biomarker inthe second biological sample of the subject is lower than the level ofthe biomarker in the first biological sample of the subject.

In certain embodiments methods of monitoring the compliance of a subjectwith a treatment of a disease, disorder, or condition with a treatmentcompound, comprise: a) obtaining a biological sample from the subject;b) determining the level of a biomarker in the biological sample,wherein the biomarker is CD44, CD83 or combination thereof, and c)comparing the level of the biomarker with the level of the biomarker ina control untreated sample from the subject; wherein the change in thelevel of the biomarker in the biological sample in comparison with thelevel of the biomarker in the control sample indicates the compliance ofthe subject with the treatment.

In certain embodiments methods of monitoring the compliance of a subjectwith a treatment of a disease, disorder, or condition with a treatmentcompound, comprise: a) obtaining a biological sample from the subject;b) determining the level of a biomarker in the biological sample,wherein the biomarker is CD83; and c) comparing the level of thebiomarker with the level of the biomarker in a control untreated samplefrom the subject; wherein an increased level of the biomarker in thebiological sample in comparison with the level of the biomarker in thecontrol sample indicates the compliance of the subject with thetreatment.

In certain embodiments methods of monitoring the compliance of a subjectwith a treatment of a disease, disorder, or condition with a treatmentcompound, comprise: a) obtaining a biological sample from the subject;b) determining the level of a biomarker in the biological sample,wherein the biomarker is CD44; and c) comparing the level of thebiomarker with the level of the biomarker in a control untreated samplefrom the subject; wherein a decreased level of the biomarker in thebiological sample in comparison with the level of the biomarker in thecontrol sample indicates the compliance of the subject with thetreatment.

In certain embodiments the treatment compound is an immunomodulatorycompound. In certain embodiments the treatment compound is3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dionehydrochloride, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof. In one embodiment,the compound is(S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

7.7 Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms provided herein comprise a compound provided herein, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,racemate, clathrate, or prodrug thereof. Pharmaceutical compositions anddosage forms can further comprise one or more excipients.

Pharmaceutical compositions and dosage forms provided herein can alsocomprise one or more additional active ingredients. Examples of optionalsecond, or additional, active ingredients are disclosed above.

Single unit dosage forms provided herein are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), topical (e.g., eye drops or other ophthalmicpreparations), transdermal or transcutaneous administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; eye drops or other ophthalmic preparations suitable fortopical administration; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms will typically varydepending on their use. For example, a dosage form used in the acutetreatment of a disease may contain larger amounts of one or more of theactive ingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Theseand other ways in which specific dosage forms are used will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 20^(th) ed., Mack Publishing,Easton Pa. (2000).

In one embodiment, pharmaceutical compositions and dosage forms compriseone or more excipients. Suitable excipients are well known to thoseskilled in the art of pharmacy, and non-limiting examples of suitableexcipients are provided herein. Whether a particular excipient issuitable for incorporation into a pharmaceutical composition or dosageform depends on a variety of factors well known in the art including,but not limited to, the way in which the dosage form will beadministered to a patient. For example, oral dosage forms such astablets may contain excipients not suited for use in parenteral dosageforms. The suitability of a particular excipient may also depend on thespecific active ingredients in the dosage form. For example, thedecomposition of some active ingredients may be accelerated by someexcipients such as lactose, or when exposed to water. Active ingredientsthat comprise primary or secondary amines are particularly susceptibleto such accelerated decomposition. Consequently, provided arepharmaceutical compositions and dosage forms that contain little, ifany, lactose other mono- or di-saccharides. As used herein, the term“lactose-free” means that the amount of lactose present, if any, isinsufficient to substantially increase the degradation rate of an activeingredient.

Lactose-free compositions can comprise excipients that are well known inthe art and are listed, for example, in the U.S. Pharmacopeia (USP)25-NF20 (2002). In general, lactose-free compositions comprise activeingredients, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. In one embodiment,lactose-free dosage forms comprise active ingredients, microcrystallinecellulose, pre-gelatinized starch, and magnesium stearate.

Also provided are anhydrous pharmaceutical compositions and dosage formscomprising active ingredients, since water can facilitate thedegradation of some compounds. For example, the addition of water (e.g.,5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf-life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed.,Marcel Dekker, NY, NY, 1995, pp. 379-80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms can be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions. Pharmaceutical compositions and dosage formsthat comprise lactose and at least one active ingredient that comprisesa primary or secondary amine are anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are, in one embodiment, packaged using materials known toprevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastics, unit dose containers(e.g., vials), blister packs, and strip packs.

Also provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. In one embodiment, dosage forms comprise a compoundprovided herein in an amount of from about 0.10 to about 500 mg. Inother embodiments, dosage forms comprise a compound provided herein inan amount of about 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50,100, 150, 200, 250, 300, 350, 400, 450, or 500 mg.

In other embodiments, dosage forms comprise the second active ingredientin an amount of 1 to about 1000 mg, from about 5 to about 500 mg, fromabout 10 to about 350 mg, or from about 50 to about 200 mg. Of course,the specific amount of the second active agent will depend on thespecific agent used, the diseases or disorders being treated or managed,and the amount(s) of a compound provided herein, and any optionaladditional active agents concurrently administered to the patient.

7.7.1 Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be provided as discrete dosage forms, such as, but not limited to,tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g.,flavored syrups). Such dosage forms contain predetermined amounts ofactive ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Oral dosage forms provided herein are prepared by combining the activeingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

In one embodiment, oral dosage forms are tablets or capsules, in whichcase solid excipients are employed. In another embodiment, tablets canbe coated by standard aqueous or nonaqueous techniques. Such dosageforms can be prepared by any of the methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is, in oneembodiment, present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants may be used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients may be used to form solid oral dosage forms. Theamount of disintegrant used varies based upon the type of formulation,and is readily discernible to those of ordinary skill in the art. In oneembodiment, pharmaceutical compositions comprise from about 0.5 to about15 weight percent of disintegrant, or from about 1 to about 5 weightpercent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel(AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), acoagulated aerosol of synthetic silica (marketed by Degussa Co. ofPlano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants may be used in an amount of less than about 1 weight percentof the pharmaceutical compositions or dosage forms into which they areincorporated.

In one embodiment, a solid oral dosage form comprises a compoundprovided herein, anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

7.7.2 Controlled Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients provided herein.Thus, the compositions provided encompass single unit dosage formssuitable for oral administration such as, but not limited to, tablets,capsules, gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in a subject at anappropriate site determined by a practitioner of skill, i.e., thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)). The active ingredient can be dispersedin a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

7.7.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. In someembodiments, administration of a parenteral dosage form bypassespatients' natural defenses against contaminants, and thus, in theseembodiments, parenteral dosage forms are sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms. For example, cyclodextrin and its derivativescan be used to increase the solubility of a compound provided herein.See, e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

7.7.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms provided herein include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th), 18^(th) and 20^(th) eds., Mack Publishing, Easton Pa. (1980,1990 and 2000); and Introduction to Pharmaceutical Dosage Forms, 4thed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedherein are well known to those skilled in the pharmaceutical arts, anddepend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied. In one embodiment,excipients include, but are not limited to, water, acetone, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof to formsolutions, emulsions or gels, which are non-toxic and pharmaceuticallyacceptable. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms. Examples of additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18^(th) and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 and 2000).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients. Also,the polarity of a solvent carrier, its ionic strength, or tonicity canbe adjusted to improve delivery. Compounds such as stearates can also beadded to pharmaceutical compositions or dosage forms to alter thehydrophilicity or lipophilicity of one or more active ingredients so asto improve delivery. In other embodiments, stearates can serve as alipid vehicle for the formulation, as an emulsifying agent orsurfactant, or as a delivery-enhancing or penetration-enhancing agent.In other embodiments, salts, solvates, hydrates, prodrugs, clathrates,or stereoisomers of the active ingredients can be used to further adjustthe properties of the resulting composition.

7.7.5 Kits

In one embodiment, active ingredients provided herein are notadministered to a patient at the same time or by the same route ofadministration. In another embodiment, provided are kits which cansimplify the administration of appropriate amounts of activeingredients.

In one embodiment, a kit comprises a dosage form of a compound providedherein. Kits can further comprise additional active ingredients such asother anti-inflammatory, immunomodulatory or immunosuppressantcompounds, or a combination thereof. Examples of the additional activeingredients include, but are not limited to, those disclosed herein.

In other embodiments, kits can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits can further comprise cells or blood for transplantation as well aspharmaceutically acceptable vehicles that can be used to administer oneor more active ingredients. For example, if an active ingredient isprovided in a solid form that must be reconstituted for parenteraladministration, the kit can comprise a sealed container of a suitablevehicle in which the active ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

8. EXAMPLES

The following Examples are presented by way of illustration, notlimitation. In the examples, test compound refers to(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

8.1 Example 1: Preparation of(S)-3-[4-(4-Morphlin-4-Ylmethylbenzyloxy)-1-Oxo-1,3-Dihydro-Isoindo-2-Yl]Piperidine-2,6-DioneHydrochloride

8.1.1 3-Hydroxy-2-methyl-benzoic acid methyl ester

3-Hydroxy-2-methylbenzoic acid (105 g, 690 mmol) was added to MeOH (800mL) in a 2 L three neck round bottom flask equipped with condenser,thermometer and stirring bar followed by the addition of MeOH (250 ml).H₂SO₄ (10 mL, 180 mmol) was added to above solution. The reactionmixture was stirred at 62° C. for 17 hours. The solvent was removed invacuo. The residue (200 mL) was added to water (600 mL) slowly at roomtemperature and a white solid was formed. The suspension was stirred inan ice bath for 30 minutes and filtered. The solid was washed with water(5×250 mL) and dried to give 3-hydroxy-2-methyl-benzoic acid methylester as a white solid (100 g, 87% yield). The compound was used in thenext step without further purification: LCMS MH=167; ¹H NMR (DMSO-d₆) δ2.28 (s, 3H, CH₃), 3.80 (s, 3H, CH₃), 6.96-7.03 (m, 1H, Ar), 7.09 (t,J=7.8 Hz, 1H, Ar), 7.14-7.24 (m, 1H, Ar), 9.71 (s, 1H, OH).

8.1.2 3-(tert-Butyl-dimethyl-silanyloxy)-2-methyl-benzoic acid methylester

To a 1 L three neck RB flask equipped with stirring bar and thermometer,were added DMF (300 mL), methyl 3-hydroxy-2-methylbenzoate (90 g, 542mmol) and imidazole (92 g, 1,354 mmol). TBDMS-Cl (90 g, 596 mmol) wasadded to the above solution in portions to control the internal tempbetween 15-19° C. over 20 minutes, and after addition, the internal tempdropped below 1° C. The ice bath was removed and the reaction mixturewas stirred at room temperature for 16 hours. The reaction mixture wasadded to ice water (500 mL), and the resulting solution was divided intotwo portions (700 mL×2). Each portion was extracted with EtOAc (700 mL).Each organic layer was washed with cold water (350 mL) and brine (350mL). Organic layers were combined and dried by MgSO₄. The combinedorganic layer was concentrated to give3-(tert-butyl-dimethyl-silanyloxy)-2-methyl-benzoic acid methyl ester asa light brown oil (160 g, 100% crude yield). The compound was used inthe next step without further purification: LCMS MH=281; ¹H NMR(DMSO-d₆) δ −0.21 (s, 6H, CH₃, CH₃), 0.73-0.84 (m, 9H, CH₃, CH₃, CH₃),2.10 (s, 3H, CH₃), 3.60 (s, 3H, CH₃), 6.82 (dd, 1H, Ar), 6.97 (t, J=7.9Hz, 1H, Ar), 7.13 (dd, J=1.1, 7.7 Hz, 1H, Ar).

8.1.3 2-Bromomethyl-3-(tert-butyl-dimethyl-silanyloxy)-benzoic acidmethyl ester

NBS (49.8 g, 280 mmol) was added to methyl 3-(tert-butyldimethylsilyloxy)-2-methylbenzoate (78.4 g, 280 mmol) in methyl acetate(500 mL) at room temperature to give an orange colored suspension. Theresulting reaction mixture was heated in an oil bath at 40° C. andshined by 300 wt sunlight bulb at reflux for 4 hours. The reactionmixture was cooled down and washed by Na₂SO₃ solution (2×600 mL, 50%saturated concentration), water (500 mL) and brine (600 mL). The organiclayer was dried by MgSO₄ and decolorized by charcoal. The organic layerwas concentrated to give2-bromomethyl-3-(tert-butyl-dimethyl-silanyloxy)-benzoic acid methylester as a light brown oil (96 g, 91% crude yield). The compound wasused in the next step without further purification: LCMS M-Br=279; ¹HNMR (DMSO-d₆) δ 0.05-0.11 (m, 6H, CH₃, CH₃), 0.82 (s, 9H, CH₃, CH₃,CH₃), 3.65 (s, 3H, CH₃), 4.74 (s, 2H, CH₂), 6.94 (dd, J=1.3, 8.1 Hz, 1H,Ar), 7.10-7.20 (m, 1H, Ar), 7.21-7.29 (m, 1H, Ar).

8.1.4 4-Carbamoyl-butyric acid methyl ester

To a stirred solution of methyl2-(bromomethyl)-3-(tert-butyldimethylsilyloxy)benzoate (137.5 g, 325mmol) in acetonitrile (1100 mL) in a 2 L round bottom flask, was addedmethyl 4,5-diamino-5-oxopentanoate hydrochloride (70.4 g, 358 mmol). Tothe suspension was added DIPEA (119 ml, 683 mmol) through an additionfunnel over 10 minutes and the suspension was stirred at roomtemperature for 1 hour before the mixture was heated in an oil bath at40° C. for 23 hours. The reaction mixture was concentrated under vacuo.The residue was stirred in ether (600 mL), and a white solidprecipitated out. The mixture was filtered and the solid was washed withether (400 mL). The filtrate was washed with HCl (1N, 200 mL),NaHCO₃(sat. 200 mL) and brine (250 mL). The aqueous acid layer and basiclayer were kept separately. Then the solid was further washed with ether(250 mL) and the liquid was washed with above acid solution and basicsolution. The two organic layers were combined and concentrated undervacuo to give4-[4-(tert-Butyl-dimethyl-silanyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-4-carbamoyl-butyricacid methyl ester as a brown oil (152 g, 115% crude yield, 77% purity byH NMR). The compound was used in the next step without furtherpurification: LCMS MH=407.

8.1.5 4-Carbamoyl-4-(4-hydroxy-1-oxo-1,3-dihydro-isoindol-2-yl)-butyricacid methyl ester

To a stirred cold solution of methyl5-amino-4-(4-(tert-butyldimethylsilyloxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate(152 g, 288 mmol) in DMF (500 mL) and water (55 mL), was added by K2CO3(19.89 g, 144 mmol) by portions over 5 minutes. The resulting reactionmixture was stirred at room temperature for 40 minutes. The reactionmixture was cooled in an ice bath. To the mixture, HCl (12M, 23.99 ml,288 mmol) was added slowly. After the addition, acetonitrile (280 mL)was added to the mixture and a solid precipitated out. The mixture wasstirred at room temperature for 10 minutes and filtered. The solid waswashed with acetonitrile (50 mL×4). The filtrate was concentrated underhigh vacuo to give a yellow oil (168 g). The oil was dissolved inacetonitrile (600 mL) and stirred at room temperature for 10 minutes.The mixture was filtered and the solid was washed with acetonitrile (25mL×2). The filtrate was concentrated under high vacuo to give a yellowoil (169 g), which was added to a mixture of water (1200 mL) and ether(1000 mL). The mixture was stirred for 3 minutes and the layers wereseparated. The aqueous solution was concentrated under high vacuo andthe residue was stirred in acetonitrile (160 mL) and a white solid wasformed after overnight stirring. The mixture was filtered to give4-carbamoyl-4-(4-hydroxy-1-oxo-1,3-dihydro-isoindol-2-yl)-butyric acidmethyl ester as a white solid (46 g, 54% yield). The filtrate wasconcentrated and the residue was further crystallized in acetonitrile(60 mL) to give more4-carbamoyl-4-(4-hydroxy-1-oxo-1,3-dihydro-isoindol-2-yl)-butyric acidmethyl ester as a white solid (11.7 g, 14% yield). The filtrate wasconcentrated and the residue was purified by ISCO chromatography to givemore 4-carbamoyl-4-(4-hydroxy-1-oxo-1,3-dihydro-isoindol-2-yl)-butyricacid methyl ester as a white solid (13.2 g, 15% yield). The totalproduct obtained was 70.9 g in 83% yield: LCMS MH=293; ¹H NMR (DMSO-d₆)δ 1.95-2.34 (m, 4H, CH₂, CH₂), 3.51 (s, 3H, CH₃), 4.32 (d, J=17.6 Hz,1H, CHH), 4.49 (d, J=17.4 Hz, 1H, CHH), 4.73 (dd, J=4.7, 10.2 Hz, 1H,CHH), 6.99 (dd, J=0.8, 7.9 Hz, 1H, Ar), 7.10-7.23 (m, 2H, Ar, NHH),7.25-7.38 (m, 1H, Ar), 7.58 (s, 1H, NHH), 10.04 (s, 1H, OH).

8.1.63-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

Step 1: To the solution of3-(4-hydroxy-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione (2.5g, 8.56 mmol) in THE (60 mL) was added triphenyl phosphine (polymersupported 1.6 mmol/g, 12 g, 18.8 mmol). The mixture was stirred at roomtemperature for 15 minutes. Diisopropyl azodicarboxylate (3.96 mL, 18.8mmol) was added at 0° C., and the mixture was stirred at 0° C. for 30minutes. (4-Morpholin-4-ylmethyl-phenyl)-methanol (2.62 g, 12.4 mmol)was added at 0° C., and the mixture was allowed to warm to roomtemperature and stirred at room temperature overnight. The reactionmixture was filtered, and the filtrate was concentrated. The resultingoil was purified on silica gel column eluted with methylene chloride andmethanol (gradient, product came out at 6% methanol) to give4-carbamoyl-4-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-butyricacid methyl ester (2.2 g, 54% yield). The product was used in the nextstep without further purification.

Step 2: To the TH solution (50 mL) of4-carbamoyl-4-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-butyricacid methyl ester (2.2 g, 4.57 mmol) was added potassium tert-butoxide(0.51 g, 4.57 mmol) at 0° C. The mixture was stirred at 0° C. for 10minutes and was quenched with 1N HCl (5 mL, 5 mmol) followed bysaturated NaHCO₃ (25 mL). The mixture was extracted with EtOAc (2×50mL). The organic layer was washed with water (30 mL), brine (30 mL),dried over MgSO₄ and concentrated. To the resulting solid was addedEtOAc (10 mL) followed by hexane (10 mL) under stirring. The suspensionwas filtered to give3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dioneas white solid (1.5 g, 73% yield). HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, gradient to 95/5 acetonitrile/0.1% H₃PO₄in 5 min: t_(R)=4.78 min (97.5%); mp: 210-212° C.; ¹H NMR (DMSO-d₆) δ1.86-2.09 (m, 1H, CHH), 2.29-2.38 (m, 4H, CH₂, CH₂), 2.44 (dd, J=4.3,13.0 Hz, 1H, CHH), 2.53-2.64 (m, 1H, CHH), 2.82-2.99 (m, 1H, CHH), 3.46(s, 2H, CH₂), 3.52-3.61 (m, 4H, CH₂, CH₂), 4.18-4.51 (m, 2H, CH₂), 5.11(dd, J=5.0, 13.3 Hz, 1H, NCH), 5.22 (s, 2H, CH₂), 7.27-7.38 (m, 5H, Ar),7.40-7.53 (m, 3H, Ar), 10.98 (s, 1H, NH)¹³C NMR (DMSO-d₆) δ 22.36,31.21, 45.09, 51.58, 53.14, 62.10, 66.17, 69.41, 114.97, 115.23, 127.64,128.99, 129.81, 129.95, 133.31, 135.29, 137.68, 153.50, 168.01,170.98,172.83; LCMS: 465; Anal Calcd for C₂₅H₂₇N₃O₅+0.86 H₂O: C, 64.58;H, 6.23; N, 9.04; Found: C, 64.77; H, 6.24; N, 8.88.

(S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dioneand(R)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dionewere prepared from3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dionethrough chiral separation.

8.2 Example 2: Effect on the Expression of Transcription Factors inPrimary Human B Cell Differentiation Model

In this example, the effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(test compound) on the expression of transcription factors controllingplasma cell differentiation, and immunoglobulin production, using an invitro human B-cell differentiation culture system.

The following abbreviations are used in this example:

Abbreviation or Specialist Term Explanation or Definition BCL6 B-celllymphoma 6 protein BLIMP-1 B-lymphocyte-induced maturation protein 1EtOH Ethanol FBS Fetal bovine serum DMSO Dimethyl sulfoxide IRF-4Interferon regulatory factor 4 MFI Mean fluorescence intensity PAX5Paired box protein Pax-5 SLE Systemic lupus erythematosus XBP-1 X-boxbinding protein 1

50 ml Buffy coat from healthy donors were obtained from Blood Center ofNew Jersey. SLE Lupus PBMC samples were obtained from Conversant Bio(Huntsville, Ala. 35806).

The following cell culture reagents were used in this study.

ITEM Source Iscoves Modified Dulbecco Invitrogen medium Fetal BovineSerum Lonza Human Insulin Sigma Human Transferrin Sigma penicillin/streptomycin Lonza Recombinant Human IL-2 R & D Systems RecombinantHuman IL-6 R & D Systems Recombinant Human IL-10 R & D SystemsRecombinant Human IL-15 R & D Systems CD40 Ligand/TNFSF5/ R & D Systemshistidine-tagged polyHistidine mouse IgG1 R & D Systems antibody ODN2006-Human TLR9 Invivogen ligand Human Interferon ALPHA A PB interferonsource

The following were used in flow cytometry analysis.

ITEM Source FITC anti-human CD19 IBD Pharmigen FITC anti-human CD20 IBDPharmigen PE anti-human CD27 IBD Pharmigen PE anti-human CD38 IBDPharmigen APC anti-human CD38 IBD Pharmigen FITC anti-mouse IgG1kIsotype IBD Pharmigen PE anti-mouse IgG1k Isotype IBD Pharmigen FITCanti-mouse IgG2bk Isotype IBD Pharmigen APC anti-mouse IgG1k Isotype IBDPharmigen Stain Buffer IBD Pharmigen

The following gene primers were used for RT-PCR:

ITEM Source AICDA gene expression assay Applied Biosystem BCL6 geneexpression assay Applied Biosystem GAPDH gene expression assay AppliedBiosystem IGJ gene expression assay Applied Biosystem IRF4 geneexpression assay Applied Biosystem PAX5 gene expression assay AppliedBiosystem PRDM1 gene expression assay Applied Biosystem XBP1 geneexpression assay Applied Biosystem Reverse Transcription Kit AppliedBiosystem Master Mix Applied Biosystem

8.2.1 Purification of hPBMCs

Fifty ml human buffy coat was aliquoted 25 ml each into two 50 mlconical tubes and 25 ml sterile HBSS was added to each conical tube. Thetubes were gently mixed by inverting. Fifteen ml of room temperatureFicoll-Paque Plus (GE Healthcare; cat #17-1440-02) was aliquoted intofour 50 ml conical tubes. Then 25 ml of the Buffy coat/HBSS mixture waslayered gently and slowly on top of the Ficoll. The samples werecentrifuged at 450 rpm for 35 minutes. The top layered containing plasmawas pipetted off and discarded. The interface containing mononuclearcells was transferred into two 50 ml conical tubes. Both conical tubeswere filled to total volume of 50 ml with HBSS and centrifuged at 1200rpm for 10 minutes. The cells were washed again in HBSS and spun at 1000rpm for 10 minutes. Cell pellet was resuspended with 20 mL of B cellmedia (Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin and countedon the cell counter.

8.2.2 B Cell Enrichment CD19+

Purified PBMCs were counted and aliquoted at 2×10⁸ cells per tube. Thecells were centrifuged at 1200 rpm for 5 minutes and then supernatantswere discarded. The cells were resuspended in 4 mL of Robosep Buffer(Stemcell Technologies catalog #20104) and transferred to a 14 mLpolystyrene round bottom tube (BD catalog #352057) and mixed well. Then200 μL of EasySep Human B cell enrichment cocktail was added (StemCellTechnologies catalog #19054). Samples were vortexed and incubated atroom temperature for 10 minutes. Next 300 μL of EasySep Magneticparticles (vortexed) (StemCell Technologies catalog #19054) were addedto the tube. Samples were vortexed and incubated at room temperature for5 minutes. After the 5 minute incubation, 5 mL of Robosep buffer wasadded to the tube and mixed well by pipetting up and down. The tube wasimmediately places in the silver magnet (StemCell Technologies catalog#19054) and incubated at room temperature for 5 minutes. Afterincubation, in one continuous motion, invert magnet and tube and pouroff desired fraction into a 50 mL conical. These procedures wererepeated for remaining PBMCs (per one donor) and combined. The combinedfraction was centrifuged at 1200 rpm for 5 minutes and then supernatantswere discarded and cells were resuspended in 5 mL of B cell media. Theisolated CD19+ cells were counted on the cell counter.

8.2.3 B Cell Differentiation Assay

Step 1—B cell Activation—day 0 through day 4: Prepare fresh B cellcocktail by adding 50 μg/mL of human transferrin to B cell media.(Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin). Filter requiredvolume of media needed for experiment through a 0.22 μM filter. Add Bcell differentiation cocktail (final concentration): recombinant humanIL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), CD40Ligand/TNFSF5/histidine-tagged (50 ng/mL), polyHistidine mouse IgG1antibody (5 μg/mL), and ODN 2006—Human TLR9 ligand (10 μg/mL) to cells.Five milliliters (1×10⁵/ml) of CD19+ B cell were added to each well of a6 well flat-bottom plate (final cell count=5×10⁵/well). Five L(lx)±compound/DMSO was added to each test well (0.1% final DMSO) andincubated at 37° C. for 4 days.

Step 2—Plasmablast Generation—day 4 through day 7: Cells were harvestedand counted on the cell counter; an aliquot was removed for flowanalysis, the remaining cells were washed with PBS. Prepare fresh B cellcocktail by adding 1 μg/ml of human transferrin to B cell media.(Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin) Filter requiredvolume of media needed for experiment through a 0.22 μM filter. Add Bcell differentiation cocktail (final concentration): recombinant humanIL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), IL-6 (50 ng/mL) tocells. Add fresh B cell cocktail and transfer cells back to the originalwells and bring volume back to 5 mL. Five L (1×)±compound/DMSO was addedto each test well (0.1% final DMSO) and incubated at 37° C. for 4 days.

On day 7, cells were harvested and counted on the cell counter. Cellswere then divided for flow analysis and the remaining cells were lysedwith RLTbuffer and stored at −80° C. for RNA extraction and geneexpression. Supernatants were aliquoted and frozen at −20° C. forimmunoglobulin assays.

8.2.4 Preparation of Test Compound Stock Solutions and Dilutions

The test compounds was weighed and dissolved in sterile 100% DMSO(dimethyl sulfoxide; Research Organics, Cleveland, Ohio) to create 40 mMstock solution. Dilutions of the 40 mM stock were used in the assay toobtain final test compound concentrations based on experimental design.

8.2.5 RNA Extraction and Gene Expression

Differentiated B cells (see paragraphs [00226]-[00227]) were harvestedfor total ribonucleic acid (RNA) preparation with a Qiacube RNAextraction instrument (Qiagen, Valencia, Calif.) using QIAGEN RNeasymini spin-column kits. Purified RNA was reverse transcribed into cDNAwith thermal cycler [MJ Research; Inc., St. Bruno, Quebec, Canada) usinga reverse-transcriptase kit (Applied Biosystems). The gene expressionassay was carried out using 7500 RT-PCR system (Applied Biosystems) intriplicate. A glyceraldehyde 3-phosphate dehydrogenase gene expressionassay control was run for each sample and used as a normalizationcontrol. For each gene, samples within each experiment were normalizedto 0.1% DMSO treatment only for that particular time point.

Supernatants (from paragraph [00228]) were harvested and analyzed byELISA for IgG and IgM production (ZeptoMetrix Corp. Buffalo, N.Y.).

8.2.6 Cell Phenotyping

Differentiated B cells (see paragraphs [00226]-[00227]) were harvested,counted, and aliquoted at about 1×10⁶ cells or less per 4 mL tube. Thecells were washed 1× with stain buffer. Next, the cells then wereblocked with 10% human serum/PBS for 20-30 minutes. Following blocking,the cells were centrifuged for 5 minutes at 1200 rpm and supernatantsdiscarded. In the 100 μL of remaining buffer, 20 μL of various BDPharmigen flow antibodies were added according to experimental design.The cells were stained for 20-30 minutes at 4° C. Then the cells werewashed 2× with stain buffer and supernatants discarded. Next, 500 μL ofstain buffer or PBS was added to the tubes. The samples were immediatelyanalyzed or put at 4° C. overnight. Cells were stained with mouseanti-human CD20 and CD38, CD19 and CD27, or respective isotype controls.All samples were analyzed using a FACSCanto flow cytometer, FACSDivaanalysis software (BD Bioscience), and FlowJo Analysis software.

8.2.7 Cell Viability Analysis

To determine live cell count, B cells (see paragraphs [00226]-[00227])were stained with 0.4% trypan blue and live cells counted using theCountess automated cell counter (Invitrogen) in duplicate samples.

The data was graphed using GraphPad Prism 5.0 software. IC₅₀ values werecalculated using non-linear regression, sigmoidal-dose responseconstraining the top to 100% and bottom to 0% allowing for a variableslope. The results for test compounds in the Ig assays were expressed asthe percentage inhibition relative to control DMSO values.

The test compound dose-dependently reduced the percentage of CD20-CD38+plasmablasts, and increased the percentage of CD20+CD38-activated Bcells. The plasmablast population (quadrant 1) was 30.4% in the DMSOcontrol at day 7, and the test compound reduced the population to 27.3%at 2 nM, 2.1% at 20 nM, and 0.4% at 200 nM (FIG. 1). The test compoundreduced B cell viability during the first 4 days of culture (FIG. 2).The effect of the test compound on B and plasma cell transcriptionfactor expression is depicted in FIG. 3. The effect of the test compoundon IgG production in plasmablast cultures is depicted in FIG. 4.

The test compound dose-dependently inhibited expression of plasma celltranscription factors RF4, BLIMP1, and XBP1 significantly. The testcompound enhanced B cell transcription factor PAX5. The effect of thetest compound on B and plasma cell transcription factor expression isdepicted in FIGS. 5A and 5B.

The test compound, pomalidomide, and lenalidomide inhibit IgG productionwith IC₅₀ of 0.0018 μM, 0.049 μM, and 0.32 μM, respectively. The dataindicated that the test compound is 27-fold more potent thanpomalidomide at inhibiting IgG production during plasmablastdifferentiation. The effect of the test compound on IgG production in Bcell cultures on days 4, 7 and 10 is depicted in FIG. 6. The effect ofthe test compound, alone and in combination with prednisolone, on IgGproduction by in vitro-differentiated plasma blasts/plasma cells isdepicted in FIG. 7. The effect of the test compound on CD20/CD38expression during B cell differentiation at day 7 is depicted in FIG. 8.The effect of the test compound on cell viability during plasmablastdifferentiation is depicted in FIG. 9.

In peripheral blood mononuclear cell isolated by patients with systemiclupus erythematosus (SLE), the test compound inhibited IgG and IgMproduction with IC₅₀s of 3.2 nM and 0.9 nM, respectively. These findingsindicated that the test compound has the potential to inhibit B celldifferentiation to the plasma cell lineage, and suggested that the testcompound may be useful in the treatment of autoimmune disorders such asSLE, which are characterized by the overproduction of autoantibodies.The effect of the test compound on B cell differentiation and functionin SLE patient PBMC cells is depicted in FIG. 10. The effect of the testcompound on IgG and IgM productions in normal and SLE patient PBMC cellsis depicted in Table 1.

FIGS. 11A and 11B depict the effect of the test compound on human IgGand IgM production, respectively, in B cell cultures on day 7.

TABLE 1 Potency for Inhibition of Normal and SLE PBMC Production of IgGand IgM IgG IC₅₀ (nM) IgM IC₅₀ (nM) SLE Normal SLE Normal (n = 3) (n =3) (n = 3) (n = 3) Test Compound 3.2 2.1 0.9 0.35 pomalidomide 19 63 3.817 apremilast >10,000 >10,000 >10,000 >10,000

8.3 Example 3: B Cell Differentiation Assay Using Flow Cytometry andLaser Scanning Cytometry

Cell Culture Materials: Enriched normal B cells and SLE patientperipheral blood mononuclear cells (PBMC) were cultured in the in vitroB cell differentiation system with IMDM medium (Invitrogen) and 10% FCS,supplemented with human transferrin and human insulin (Sigma) pluscytokine cocktail. Test compound was added to culture on day 0 and day4.

B Cell Differentiation Protocol: Enriched B cells were isolated fromfresh buffy-coat (leukocyte enriched units) by Ficoll-Hypaque densitygradient centrifugation followed by incubation with EasySep negativeselection human B cell enrichment kit (Stem cell technologies). Inbrief, 2×10⁸/ml PBMC were mixed with 4 ml of Robosep buffer andtransferred to a 14 mL polystyrene round bottom tube. EasySep Human Bcell enrichment cocktail (200 μL) was added per tube, vortexed andincubated at room temperature for 10 minutes. EasySep Magnetic particleswere added (300 μL per tube) and vortexed and incubated at roomtemperature for 5 minutes. Robosep buffer (5 mL) was added to each tubeand mixed well by pipetting up and down. Tubes were placed in the silvermagnet and incubated at room temperature for 5 minutes. The magnet andtube were picked up and in one continuous motion inverted, pouring offthe desired fraction into a 50 mL conical. Cells were spun at 1200 rpmfor 5 minutes. Supernatant was poured off and 5 mL of fresh B cell mediaadded. After cells were counted, an aliquot was removed for FACSanalysis and the remaining cells were used for culture. CD19+ B cellswere isolated to ˜95% purity as determined by flow cytometry. Purified Bcells were plated at 1×10⁵ cell/ml in a sterile 6 well plate at 5 ml perwell. All cell cultures were performed in IMDM medium (Invitrogen) and10% FCS, supplemented with human transferrin and human insulin (Sigma).B-cell activation, PB generation and PC generation were performed basedon the modified in vitro system of differentiation of B cells intoplasma cells. All recombinant human cytokines IL-2, IL-4, IL-6, IL-10,IL-15, INF-α, and CD40 L and anti-polyhistidine mAb were added atindicated culture steps. Various concentrations of test compound (2, 20and 200 nM) were added to culture on day 0 and day 4. On day 4, pool allcells together from same treatment, count cells, remove cells for FACSanalysis and cytospin preparation. Plate the remaining cells at 2.5×10⁵cell/ml in a sterile 6 well plate at 5 ml per well. SLE PBMCs werecultured for 7 days under conditions to promote plasma celldifferentiation as normal B cells.

Immunophenotyping For Flow Cytometric Analysis: Cells were stained usingmulticolor direct immunofluorescence stain for flow cytometric analysis.Surface staining was performed before cell fixation andpermeabilization. Cells (50 μl; 1×10⁶ cells/ml in washing buffer, 2% FBSwith 0.1% NaN3 in PBS) were used for each staining. The cells werestained with isotype control mAb (1 g/106 cells) and multicolorFITC-conjugated anti-CD20 mAb and PE-conjugated anti-CD38 mAb, orPerCP-Cy5.5 conjugated anti-CD44 mAb, PE-conjugated anti-CD83 mAb wereused for day 4 activated B cells (CD20+CD38-cells), and day 4 PBs andday 7 PBs (CD20-CD38+), and other staining, analyzed by flow cytometryas per manufacturer's instructions. The intracellular staining oftranscription factor proteins (BCL-6, IRF-4, BLIMP-1, PAX-5 and XBP-1)and IgJ was performed according to the manufacturer's recommendations.Flow cytometric analysis was performed with a FACSCanto using FACSDiva 6(BD Biosciences). For data analysis, Flowjo (Tree Star) software wasused.

Preparation Of Cytospin From Single Cell Suspension AndImmunofluorescence Staining for iCyte: A cell suspension of not morethan 0.5×10⁶ cells/ml of 2% FBS-containing PBS was prepared. Up to 200μl of this suspension was loaded in each cuvette and spinned at 800 rpmfor 3 min. The cuvette and the paper were carefully detached withoutdamaging the fresh cytospin and proceeded with either immediate fixationor drying. Unfixed cytospins were stored for max 2 days at roomtemperature. Cells on slides were fixed with increasing concentrationsof EtOH and allowed to dry prior to staining. After blockingnon-specific binding with normal serum (species same as secondaryantibody), sections were incubated with the mixture of followingmonoclonal antibodies in a humidified chamber at cool room forovernight: (i) anti-human CD38 as plasmablast cell marker, and (ii)anti-BCL-6, IRF-4, BLIMP-1, PAX5 and XBP-1 for transcription factorproteins. After washings, slides were incubated for 1 hr at roomtemperature in dark with the mixture of secondary antibodies which wereraised in different species (with two different fluorochromes, i.e.,Alexa Fluor-488 and Alexa Fluor-633). The slides were then rinsed inPBS, counterstained with DAPI for 20 minutes at room temperature,coverslipped with anti-fade fluorescent mounting medium, and sealed withnail polish. The slides were stored in dark at 4° C. To exclude falsepositives produced by nonspecific binding of the secondary antibody, allof the tissues were treated in the same manner with buffer substitutingfor the primary antibody. The color of the antibody staining wasobserved and quantity was analyzed in the tissue sections using iCyte.

Laser Scanning Cytometer Image Capturing And Fluorescence QuantitativeAnalysis: Dual-color immunofluorescence stain was performed on cellswhich were cytospined on slides (Cytospin 4, Thermo scientific) based onstandard IHC method. Image capturing and fluorescence quantitativeanalysis were performed using a laser scanning cytometry (iCysquantitative imaging cytometry, CompuCyte). Two passes were set up(first pass for 488 and second pass for 405/633). Low resolution scanwas used for mosaic scan, and high resolution scan for region scan andanalysis. In brief, the slide was placed on the LSC stage and a regionselected for scanning. The LSC utilized the argon laser operating at 5mW. The slide was scanned using the 20× objective. Cells were identifiedand selected by contouring on blue fluorescence and a minimum cell size,as determined by DAPI staining. The cell detection threshold was set toselect single cells based on forward angle light scatter displayed in adot plot of cell area vs. forward scatter integral. Laser light-scatterevents were captured and used to contour single cells within the scandata display. Contour discrimination was set from the nuclear portion ofthe scanned cell such that about 67% of the total light scatterpixelation was contoured. Green and long red PMT-detector gain voltagesare set so that no greater than 75% maximum saturation of the max pixelin the respective fields is achieved during the scan. After establishingthe scan area, the slide was analyzed using a 40× objective. A minimumof 6 scanning areas for each slide was examined. A cell gallery wascreated by relocation of cells from each of the major peaks in thehistogram of integrated long red fluorescence. The morphologiccomposition of relocated cells was examined for purposes of qualityassurance. Data can be analyzed in either integrated log fluorescencemode or in linear max pixel mode. Different cell population in LSCscanning was scored as percentage and MFI, and quantitative comparisonto DMSO controls.

Data Analysis: Flow cytometry surface and intracellular molecules wereanalyzed by FACSCanto. Data analysis was done by Flowjo. The color ofthe antibody staining was observed and quantity was analyzed in thetissue sections using iCyte. Different cell population in FCM and LSCscanning was scored as percentage and MFI, and quantitative comparisonto DMSO controls. The data was expressed as Mean SEM. All of the datawas graphed using GraphPad Prism 6.1 (GraphPad Software; San Diego,Calif.). Statistical analysis was performed using One-Way ANOVA(Dunnett's multiple comparison test), and paired student t test. Pvalues less than or equal to 0.05 were considered significant.

B cells were cultured using the modified in vitro system fordifferentiation of B cells into plasma cells as described above.Multicolor FITC-conjugated anti-CD20 mAb and PE-conjugated anti-CD38 mAbwere used for day 4 activated B cells (CD20+CD38-cells) and day 7plasmablasts (PBs) (CD20-CD38+). Data from 3 separate experimentsrepresentative of 3 donors are shown in Table 2. The results showed thetest compound had a significant effect on B cell differentiation to theplasmablast/plasma cell lineage. It increased activated B cells andreduced plasmablasts, also reduced cell viability over time (FIGS. 12Aand 12B). The activated B cells (CD20+CD38-cells) treated with the testcompound (20 nM) did not change significantly between Day 4: (57.9%11.5%) and Day 7 (50.5%±8.6%), but the test compound significantlyincreased activated B cells when compared with the vehicle (DMSO)control which at Day 4 (42.1% 1.5%, p<0.05) and at Day 7 (12.5%±5.7%,p<0.05). Meanwhile, the test compound significantly reducedplasmablasts/plasma cells (CD20-CD38+) at Day 4 (4.8% 2.3%) and at Day 7(9.7% 5.4%) compared with DMSO control (Day 4, 25.9% 2.4% p<0.05; and atDay 7, 54.8% 5.0%, p<0.001). Furthermore, the test compounddose-dependently depleted large size cell population (gate p2) in thenormal B cell differentiation assay (FIG. 13). The percentage of largesize cells at Day 4 of the test compound treated was 31.2%, 23.1% and20.4% for 2 nM, 20 nM and 200 nM treatments respectively. The percentageof large size cells at Day 7 of the test compound treated was 34.9%,19.9% and 11.94% for 2 nM, 20 nM and 200 nM treatments respectively.Comparison with DMSO control, the number was at 35.5% and 34.9% at Day 4and Day 7 respectively.

TABLE 2 Test compound inhibits differentiation of plasmablasts Day 4 Day7 DMSO Test compound DMSO Test compound CD20−CD38++ 25.9 ± 2.4  4.8 ±2.3 54.8 ± 5.0  9.7 ± 5.4 plasmablasts/plasma cells, % CD20+CD38+intermediated 14.7 ± 3.1 18.3 ± 5.5 22.8 ± 5.1   23 ± 7.7 cells, %CD20+CD38− activated B cells, % 42.1 ± 1.5 57.9 ± 11.5 12.5 ± 5.7 50.5 ±8.6

The effect of the test compound on Bcell and plasma cell transcriptionfactor (BCL-6, IRF-4, BLIMP-1, PAX5 and XBP-1) expression was evaluatedby flow cytometric method. B cells were cultured as described in aboveand cells were harvested at day 4, day 7 for immunofluoresence staining.The cells were first stained for CD20 and CD38, and after cellpermeabilization, stained for BCL-6, IRF-4, BLIMP-1, PAX5 and XBP-1,then were analyzed by gating on whole lymphocytes. Data from 3experiments representative of 3 are shown in FIG. 14. The resultsindicated that the test compound (20 nM) caused a shift in transcriptionfactor expression in plasmablasts/plasma cells. It significantlydecreased IRF-4 (p<0.5), BLIMP-1 (p<0.05), and XBP-1 (p<0.05) expressionat Day 4, but significantly increased BCL-6 (p<0.05) on Day 7.

Laser scanning cytometry (iCyte) was used for quantitative analysis toconfirm the flow cytometry results described above. The B cells fromnormal donors and PBMC from SLE patients were cultured as described inabove method. The cells were harvested at Day 4 and Day 7 forcytospining cells to slides, then followed double-immunofluorescencestain as described in Methods. CD38 (green) was expressed byplasmablasts/plasma cells, transcription factor (Red) BCL-6, IRF-4,BLIMP-1, PAX5, or XBP-1 was colocalized in cytoplasm or nucleus. Thenucleus were counterstained with DAPI (blue). In Day 7 normal B cellsamples, when compared with DMSO control, the test compound (20 nM)significantly increased BCL-6 (FIG. 15A, p<0.01), decreased IRF-4 (FIG.15B, p<0.001) and BLIMP-1 (FIG. 15C, p<0.01) expression in wholelymphocyte population, and significantly increased BCL-6 (p<0.05),decreased IRF-4 (p<0.001), BLIMP-1 (p<0.001), and PAX-5 (FIG. 15D,p<0.05) expression in CD38+ plasmablast/plasma cells. There was nochange of XBP-1 (FIG. 15E) in both cell populations. In SLE patientPBMC, three transcription factors were tested: BCL-6, IRF-4 and BLIMP-1.The data indicated that the test compound had similar activity in thesetranscription factors as with healthy donor cells. The test compounddose-depend significantly increased BCL-6 expression, and inhibited RF-4and BLIMP-1 expression in CD38+ plasmablast/plasma cells fromdifferentiating Day 4 and Day 7 SLE patient PBMC (FIG. 16).

Using the same B cells in vitro differentiation system, the activity ofthe test compound on CD44 and CD83 expression in B cell differentiationwas further investigated. Multicolor FITC-conjugated anti-CD20 mAb,PerCP Cy5.5-conjugated anti-CD44 mAb and PE-conjugated anti-CD83 mAb areused for day 4 and day 7 cells. The effect of the test compound wasevaluated from 3 separate experiments representative of 3 donors. Thedata indicated that the test compound had dose-dependently andsignificantly decreased CD44 mean fluorescence intensity (MFI) (FIG. 17,p<0.01) in Day 7 B cell differentiation samples, which was due todepletion of CD20^(high)/CD44^(high) cells (FIG. 18). The percentage ofCD20^(high)/CD44^(high) cells at Day 7 of the test compound treatment at2 nM, 20 nM and 200 nM was 18.4%, 10.1% and 6.6% repetitively, comparedwith the DMSO control of 22.2%. Depletion of CD44+ cells may reduceleukocyte adhesion. The test compound also significantly increased totalCD83+ cell population and enhance CD83+ expression (FIG. 19). Thepercentage of CD83+ cells at Day 4 of the test compound treatment of 2nM, 20 nM and 200 nM was 24.1%±2.1%, 40.2%±3.6%, 49.5%±4.4% and 18.4%.The percentage of CD83+ cells at Day 7 of the test compound treatment of2 nM, 20 nM and 200 nM was 16.3%±3.3%, 36.1%±3.4% and 51.9%±0.5%,compared with DMSO at 13.4%±2.4% and 12.9%±3.3 at Day 4 and Day 7respectively.

High level IgJ chain expression in rheumatoid arthritis (RA) patientspredicts lack of response to rituximab. In order to evaluate if the testcompound has any activity on IgJ production, the cells were also usedfor intracellular staining for IgJ. The results from three donorsindicated that the test compound dose-dependently and significantlyreduced Ig J chain expression in Day 4 B cell differentiation cultures.It not only reduced the number of IgJ-positive cells (Day 4, 20 nM at8.4%±1.5%, 200 nM at 5.4%±1.8%; Day 7, 20 nM at 13.2%±2.3% and 200 nM at7.2%±1.3, respectively, compared with DMSO control at 13.1%±1.5% and14.4%±4.3 at Day 4 and Day 7 respectively), but also decreased the MFIof IgJ within the positive cells (FIG. 20). Reduced IgJ and IgG, IgMproduction may be a potential marker of response to the test compound indiseases like RA.

8.4 Example 4: Effect of Test Compounds on Cytokine and ChemokineProduction in Anti-Human CD3-Stimulated Human T Cells

This example demonstrates the effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione,(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneand3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cytokine and chemokine production in anti-human CD3-stimulated humanT cells using multiplex Luminex Technology.

The following abbreviations are used:

-   -   Abbreviation Explanation or Definition    -   IL Interleukin    -   G-CSF Granulocyte Colony Stimulating Factor    -   GM-CSF Granulocyte Macrophage Colony Stimulating Factor    -   IFN-γ Interferon Gamma    -   TNF-α Tumor Necrosis Factor Alpha    -   RANTES Regulated on Activation, Normal T Cell Expressed and        Secreted

The following materials were used in this study:

-   -   RPMI-1640 Media supplemented with 10% FBS, 100 units/mL        penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine (Life        Technologies)    -   RosetteSep® Human T-Cell Enrichment Cocktail (StemCell, Cat        #15061)    -   Luminex Human Cytokine/Chemokine 12-Plex Kit (Millipore, Cat        #MPXHCYTO-60K-12)    -   Luminex IS100 instrument (Millipore)    -   Anti-human CD3 antibody, OKT3 clone (eBioscience, Cat        #16-0037-85)

The test compounds were prepared as stock solutions of 4 mM in DMSO. Tcells were isolated from buffy coat by negative selection using theRosetteSep® T Cell Enrichment Cocktail according to manufacturer'sprocedures.

All 96-well plates were pre-coated with 3 g/mL anti-human CD3 antibodyin 100 μL 1×PBS for 4 hours at 37° C. The plates were washed 3 timeswith RPMI-1640 Complete Media prior to the T cell assay. The T cellswere then plated in anti-CD3-pre-coated plates at a density of 2.5×10⁵cells/well in 180 μL RPMI-1640 Complete Media. The cells were treatedwith 20 μL 10× titrated test compounds at 10, 1, 0.1, 0.01, 0.001,0.0001, and 0.00001 μM in duplicate. The final DMSO concentrations were0.25%. The plates were incubated for 48 hours at 37° C., 5% CO₂.

After 48 hours, the supernatants were harvested and tested by amultiplex cytometric bead array (CBA) assay for the followingcytokines/chemokines: IL-2, IL-3, IL-5, IL-10, IL-13, IL-15, IL-17A,GM-CSF, G-CSF, IFN-γ, TNF-α, and RANTES.

The CBA plates were analyzed on the Luminex IS100 instrument.

Data from each donor was graphed using GraphPad Prism 5.0 software andexpressed as mean pg/mL±SEM and % of DMSO control±SEM.

The test compounds demonstrated immunomodulatory activity in anti-CD3stimulated primary human T cells, altering the production of severalcytokines and chemokines. Baseline levels of cytokines and chemokinesproduced by stimulated human T cells incubated with vehicle arepresented in Table 3 below.

TABLE 3 Baseline levels of cytokines and chemokines Baseline AmountProduced Cytokine/Chemokine (pg/mL) IL-2 31 IL- 38 IL-5 27 IL-10 449IL-13 205 IL-17A 19 GM-CSF 132 IFN-γ 1271 TNF-α 411 RANTES 314

The test compounds enhanced IL-2, IL-3, IL-5, IL-10, IL-13, GM-CSF,IFN-γ, RANTES, and TNF-α production in stimulated human T cells. Theenhancement of production by test compounds was largelyconcentration-dependent for most of the cytokines and chemokines, exceptfor IL-10 and IL-5. The test compounds enhanced IL-10 production atlower concentrations but inhibited enhancement of IL-10 production athigher concentrations. The test compounds enhanced IL-5 productionprimarily at a single concentration within the range of concentrationsthat increased production of other cytokines. Relatively small amountsof IL-2, IL-3, IL-5, and IL-17A were produced in control cells incomparison with other cytokines and chemokines (Table 1). Production ofIL-17A did not change much by test compounds. Measurable quantities ofGCSF and IL-15 were not produced in stimulated human T cells. The effectof3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cytokine and chemokine production in anti-CD3-stimulated human Tcells, expressed as absolute amount produced and as percentage ofvehicle control cells are provided in FIGS. 21 and 22, respectively. Theeffect of(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cytokine and chemokine production in anti-CD3-stimulated human Tcells, expressed as absolute amount produced and as percentage ofvehicle control cells are provided in FIGS. 23 and 24, respectively. Theeffect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneon cytokine and chemokine production in anti-CD3-stimulated human Tcells, expressed as absolute amount produced and as percentage ofvehicle control cells are provided in FIGS. 25 and 26, respectively. Thedashed line denotes the level equivalent to double the baselineproduction (EC₂₀₀) in FIGS. 22, 24 and 26.

8.5 Example 5: Anti-Inflammatory Activity

Anti-inflammatory activities of3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione,(R)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dioneand(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionewere studied in human peripheral blood mononuclear cells (hPBMC).Luminex Technology was used to determine the inhibitory (enhancement)concentration, IC₅₀ for the compounds for the simultaneous profiling ofpro-inflammatory cytokines/chemokines and IL-10 (anti-inflammatorycytokine) from LPS-stimulated healthy human donor PBMCs.

50 ml Buffy coat from healthy donors was obtained from Blood Center ofNew Jersey (East Orange, N.J.). Lipopolysaccharide (strain)(Cat #L-1887)was purchased from Sigma. Milliplex kits with antibody bound beads forLuminex xMAP Technology was purchased from Millipore (Billerica, Mass.)and combined into multiplex format prior to assay.

8.5.1 Purification of Human Peripheral Blood Mononuclear Cells

50 ml human buffy coat was aliquoted 25 ml each into two 50 ml conicaltubes and 25 ml sterile HBSS was added to each conical tube. The tubeswere gently mixed by inverting. Fifteen ml of room temperatureFicoll-Paque Plus (GE Healthcare (location); cat #17-1440-02) wasaliquoted into four 50 ml conical tubes. Then 25 ml of the Buffycoat/HBSS mixture was layered gently and slowly on top of the Ficoll.The samples were centrifuged at 450 rpm for 35 minutes. The top layeredcontaining plasma was pipetted off and discarded. The interfacecontaining mononuclear cells was transferred into two 50 ml conicaltubes. Both conical tubes were filled to total volume of 50 ml with HBSSand centrifuged at 1200 rpm for 10 minutes. The cells were washed againin HBSS and spun at 1000 rpm for 10 minutes. Cell pellet was resuspendedwith 20 ml RPMI complete medium (RPMI/5% human sera/1× pen/strep/glut)and counted.

8.5.2 Treatment of Human Peripheral Blood Mononuclear Cells

One hundred μl (2×106/ml) of hPBMCs were added to each well of a 96 wellflat-bottom plate (final cell count=2×105/well) and incubated at 37° C.for 1 hour. Twenty (10×) compound was added to each test well and twentyμl medium containing 2.5% DMSO was added to each control well([DMSO]final=0.25%) and plate was incubated for 1 hour at 37° C. Cellswere then stimulated with 80 of 2.5 ng/ml LPS ([LPS]final=1 ng/ml) andincubated for 18 hours at 37° C.

50 μl supernatant from each well was transferred into 3 newround-bottomed 96 well plates and stored at −20° C. for Luminexanalysis. Duplicate wells were performed for each sample.

8.5.3 Luminex Analysis

Supernatant samples were analyzed for cytokines in multiplex formataccording to the manufacturer's instructions (Millipore, Billerica, Ma01821) using a Luminex IS100 instrument. IL-12 and GM-CSF analyses weredone in a two-plex format using neat supernatants while all othercytokines were done in a multiplex format using supernatants diluted1:20. Data analysis was performed using Upstate Beadview software. IC₅₀swere calculated using non-linear regression, sigmoidal dose-response,constraining the top to 100% and bottom to 0%, allowing variable slope.The EC₅₀s were based on the upper constraint of the sigmoidal curvesequaling 246.9%, representing the average IL-10 enhancement produced bypomalidomide (control) at 10 μM and the lower constraint to 100%. TheIC₅₀ were performed using GraphPad Prism v5.00. The data valuesrepresent the mean±SEM (standard error of the mean) of n (number ofexperiments in duplicate).

As demonstrated by data in Table 4 below and FIGS. 27, 29 and 31, testcompounds have varied potencies for the inhibitions of the multiplecytokines examined, e.g., Il-6, IL-8, IL-10, GM-CSF, MDC, MIP-1α,MIP-1β, and TNF-α, in general. Also, these compounds enhanced productionof IL-10, MCP-1, and RANTES with various potencies as provided in Table5 and FIGS. 28, 30 and 32.

TABLE 4 Summary of Cytokine Inhibitory Profile of Test CompoundsRacemate R-enantiomer S-enantiomer Cytokine IC₅₀ (μM) IC₅₀ (μM) IC₅₀(μM) IL-6 0.01 0.083 0.0038 IL-8 >10 >10 >10 IL-1β 0.00085 0.00620.00046 GM-CSF 0.0092 0.039 0.0022 MDC 0.0026 0.012 0.0021 MIP-1α 0.190.45 0.028 MIP-1β >10 >10 >10 TNF-α 0.0018 0.0095 0.00059

TABLE 5 Cytokine Profile Summary of-Mean % of Control at 0.1 μM RacemateR-enantiomer S-enantiomer Cytokine (% of control) (% of control) (% ofcontrol) IL-10 371 442 379 MCP-1 208 223 233 RANTES 153 151 153

8.6 Example 6: Effect on Human Natural Killer (NK) Cell Function inResponse to Igg/Rituximab

In this example, the capacity of test compounds to enhance human NK cellfunction in response to IgG/Rituximab was studied. The immunomodulatoryactivity of the test compounds was compared in two assays of naturalkiller (NK) cell functions (1) IgG- and IL-2-induced interferon-gamma(IFN-γ) production.

The materials used in the study and their sources are provided below:

-   -   Buffy Coat from healthy volunteers (Blood Center of New Jersey)    -   Ficoll-Hypaque Plus (Fisher Scientific Co LLC, PA, Cat        #17144002)    -   RPMI-1640 Medium supplemented with 10% FBS (fetal bovine serum),        100 units/mL penicillin,    -   100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen, Cat        #21870-076)    -   RPMI-1640 Medium (without phenol red) supplemented with 10% FBS,        100 units/mL penicillin,    -   100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen, Cat        #11835-030)    -   Rituximab (Rituxan, Roche, Inc.) (Cat No. DIN 02241927, Lot No.        B50177)    -   Human AB+ serum (Gemini Bio Products, CA, Cat #100-512)    -   CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, WI,        Cat #G1780)    -   RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell        Technologies, Vancouver, BC, Cat #15065)    -   Mouse anti-human CD56+ conjugated APC (BD Biosciences, CA, Cat        #555518)    -   Human Immunoglobulin G from Serum (IgG) (Sigma, St. Louis, Mo.;        Cat #I2511-10MG)    -   Human Recombinant IL-2 (R&D Systems, MN, Cat #202-IL-050/CF)    -   Human IFN-gamma ELISA Kit (ThermoFisher, Cat #PIEHIFNG5)

The following cell lines were used:

-   -   Activated B cell-like—diffuse large B cell lymphoma (ABC-DLBCL):        Riva cells (NCI, MD)    -   Germinal center B-cell-like—diffuse large B cell lymphoma        (GCB-DLBCL):    -   WSU-DLCL2 (Celgene Signal, CA)    -   Farage (ATCC, VA)    -   Follicular lymphoma: DoHH2 (DSMZ, Germany)    -   Burkitt's lymphoma (BL): Raji (ATCC, VA).

NK cells from healthy donors were isolated from buffy coat blood bynegative selection using the RosetteSep NK cell enrichment cocktail(Stem Cell Technologies, Vancouver, BC) prior to Ficoll-Hypaque (FisherScientific Co LLC, PA) density gradient centrifugation following themanufacturers' instructions. CD56+NK cells were isolated to ˜85% purity,as determined by flow cytometry (BD Biosciences, CA).

8.6.1 NK IgG-Induced Interferon-Gamma (IFN-Gamma) Assay

Ninety-six-well flat-bottom plates were coated with 100 μg/mL of humanIgG (Sigma) overnight at 4° C. The next day, unbound IgG was washed awaywith cold 1×PBS. NK cells were then plated in the IgG-coated 96-wellplates at 2×105 cells per well in 180 μL RPMI-1640 Media and 10 ng/mL ofrhIL-2 (R & D Systems, MN) was added. Test compounds were added in avolume of 20 μL DMSO. Final concentrations of test compounds were0.0001, 0.001, 0.01, 0.1, 1, or 10 μM. Final DMSO concentrations were0.25%. After 48 hours, the supernatants were harvested and analyzed byELISA for IFN-γ production.

Data used to determine the ability of test compounds to enhance NK cellIFN-γ production in response to immobilized IgG and rhIL-2 stimulationwas analyzed for each donor using GraphPad Prism v5.0 software. The dataare presented in two ways, (1) as the absolute amount if IFN-γ produced(pg/mL±SEM) and (2) as the percentage of the amount of IFN-γ produced inthe presence of 1 μM pomalidomide. The EC₅₀ is the concentration of testcompound providing half-maximal IFN-γ production, with maximalproduction defined as the amount of IFN-γ produced in the presence of 1μM pomalidomide. EC₅₀ values were calculated using non-linearregression, sigmoidaldose-response constraining the top to 100% andbottom to 0% allowing for a variable slope.

TABLE 6 Compound EC₅₀ Racemate 0.037 μM R-enantiomer 0.016 μMS-enantiomer 0.012 μM

The test compounds enhanced NK cell IFN-γ □production in a dosedependent manner in response to immobilized IgG and IL-2 stimulation.Results for the racemate, R-enantiomer and S-enantiomer are provided inFIGS. 33-35 (expressed as pg/mL of IFN-γ produced), respectively. FIGS.36-38 provide results expressed as a percentage of increased IFN-γproduced relative to the IFN-γ produced in the presence of pomalidomideat 1 μM for the racemate, R-enantiomer and S-enantiomer, respectively.Each value plotted in FIGS. 33-38 represents the mean of 12-14determinations SEM.

8.7 Example 7: Human Vascular Endothelial Cell Proliferation, TubeFormation, Migration, and Invasion Assays

In this example, racemate refers to3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione,R-enantiomer refers to(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneand S-enantiomer refers to(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione.

Human Umbilical Vascular Endothelial Cells Proliferation Assay: Humanumbilical vascular endothelial cells were thawed and grown in EGM2medium until passage 3 to 6 for all proliferation assays. Humanumbilical vascular endothelial cells were trypsinized, washed with 20%FBS/M199 medium and plated with the same medium at 104 cells/100 μL perwell to 96-well cell culture plates. The plates were incubated overnightat 37° C. to allow cells to adhere. The cells were then starved in 1%FBS/M199 medium for 18 hours after washing with the same medium 3 times.For optimization of the concentration of the growth factors in the HUVECproliferation assay, 100 μL/well of the 2× serial diluted growthfactors, starting at 100 ng/mL, were added to HUVECs in duplicate for 72hours at 37° C. in a humidified cell culture incubator with 5% CO2. Foranalysis of test compounds, a serial dilution of the test compounds in0.4% DMSO/1% FBS/M199 medium in duplicate was made from the 10 mM stock.Fifty microliters per well of the serially diluted test compounds (10,1.0, 0.1, 0.01, 0.001, 0.0001, 0.00001 μM) were added to the cells for 1to 2 hours at 37° C. The final DMSO concentration in the cells is 0.1%.Then 50 μL of 4× final concentration of relative growth factors wasadded to each well in duplicate for 72 hours at 37° C. in a humidifiedcell culture incubator with 5% CO2. Thymidine incorporation was measuredby adding one microcurie of 3H-thymidine (Amersham) in 20 μL medium toeach well and incubated at 37° C. in a humidified cell culture incubatorwith 5% CO2 for 5 to 6 hours. The cells were then trypsinized andharvested onto UniFilter GF/C filter plates (Perkin Elmer) by using thecell harvester (Tomtec). After the plates were air dried, 20 L/well ofMicroscint 20 (Packard) was added then the plates were analyzed inTopCount NXT (Packard). Each well was counted for one minute. Theexperiments were performed in duplicate in each of 3 donors.

Human Umbilical Vascular Endothelial Cell Tube Formation Assay:Compounds were tested in the growth factor-induced HUVEC tube formationassay. The tube formation plates were incubated at 37° C. for 30 minutesfor matrigel to polymerize. The HUVECs were starved in 0.1% BSA basalEBM2 medium for 5 hours after washing with the same medium 3 times. Thecells were trypsinized and centrifuged. Then 25 μL of 4× seriallydiluted compounds (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 μM) wereadded in duplicate with 50 μL of 2×104 cells/well to tube formationplates coated with matrigel. Fifty L of 4×VEGF (final concentration=25ng/mL) or bFGF (final concentration=10 ng/mL) were added to the plates.The cells were then incubated overnight (˜18 hours) at 37° C. in ahumidified incubator. The tubule webs were stained with calcein AM at 4μg/μL in 2% FBS/HBSS for 30 minutes and images taken by fluorescencemicroscopy. The tubules were quantified by the MetaMorph tube formationsoftware program for tube area and tube length.

Human Umbilical Vascular Endothelial Cell Invasion Assay: In the HUVECinvasion assay, the concentration of human fibronectin is optimized toprovide a suitable protein structure for adherent cells to attach to themembrane and allow free migration in response to an angiogenic stimulus(e.g. VEGF, bFGF, or HGF) in the lower chamber of the insertplate.HUVECs were starved in 0.1% BSA EBM2 medium for 6 hours after washingwith the same medium 3 times. The cells were then trypsinized andcentrifuged to remove the remaining trypsin. Then −0.5 to 1×106 cells in125 μL/well and 125 μL of 8× serially diluted compounds (10, 1, 0.1,0.01, 0.001 μM) were added to the upper chamber of the BD Falcon 24-welland 96-well insert plates in duplicate and incubated for −1 to 2 hours.(The plates contain a fluorescence blocking, microporous [3.0 μm poresize] PET membrane that has been evenly coated with human fibronectin.)Seven hundred fifty microliters of a 1.33×stock solution of VEGF (finalconcentration of 25 ng/mL), bFGF (final concentration of 10 ng/mL), orHGF (final concentration of 25 ng/mL) were then added to the lowerchamber. The cells were incubated for 22±1 hours at 37° C. The migratedcells were stained with calcein AM at 4 g/mL in HBSS containing 2% FBS,using 500 μL/well in 24-well plates and 200 μL/well in 96-well plates.The plates were incubated at 37° C. for 90 minutes and read in afluorescence plate reader.

The percentage inhibition of cell proliferation, tube formation,migration, and invasion was calculated by subtracting the result forunstimulated DMSO control from test sample results, averaging allreplicates, and normalizing to the growth factor-stimulated DMSO control(0% inhibition). The IC50 values were calculated by using GraphPad Prism5.0.

Human Umbilical Vascular Endothelial Cells Proliferation Assay Results:Results from the growth factor optimization study indicated that theoptimal concentrations of VEGF, bFGF, and HGF for induction ofproliferation were 25, 10, and 25 ng/mL respectively. The test compoundswere examined with optimized growth factor concentrations and resultsindicated that the racemeate, S-enantiomer, and R-enantiomer did notinhibit VEGF-, bFGF-, or HGF-induced HUVEC proliferation (FIG. 39).However, there was a significant enhancement of proliferation observedin the VEGF- and HGF-treated HUVECs by S-enantiomer (VEGF-treated: 1-10μM; HGF-treated: 0.1-1 μM). Also there was a significant enhancementobserved in the bFGF-treated HUVECs by racemate (0.01-1 μM), andR-enantiomer (0.1-1 μM). IC₅₀ values are summarized in the Table 7.

TABLE 7 Summary of IC₅₀ Values from Growth Factor-induced HumanUmbilical Vascular Endothelial Cell Proliferation Studies VEGF (25ng/mL) bFGF (10 ng/mL) HGF (25 ng/mL) IC₅₀ IC₅₀ IC₅₀ Test CompoundsValues (μM) Values (μM) Values (μM) Racemate >100 99 24R-enantiomer >100 76 38 S-enantiomer >100 52 51

Human Umbilical Vascular Endothelial Cell Tube Formation Assay Results:The test compounds displayed a trend toward inhibiting VEGF-inducedHUVEC tube formation in terms of both tube length and tube area (FIG.40). All compounds demonstrated a dose-dependent effect on VEGF-inducedHUVEC tube formation.

R-enantiomer showed significant inhibition (p<0.05 vs stimulated DMSOcontrol) of tube area and length at 10 μM. There was also a trend forthe compounds to inhibit bFGF-induced HUVEC tube formation in terms ofboth tube length and tube area (FIG. 40), although the effect was lesspronounced than the effects on VEGF-induced HUVEC tube formation.

Human Umbilical Vascular Endothelial Cell Invasion Assay Results:3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione,(R)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dioneand(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionesignificantly inhibited VEGF-, bFGF-, and HGF-induced HUVEC invasion ina dose-dependent manner (FIG. 41). The compounds were more potentagainst VEGF- and bFGF-induced HUVEC invasion than against HGFinducedHUVEC invasion (Table 8). The IC₅₀ value was <0.3 nM for inhibition ofVEGFinduced HUVEC invasion by the test compounds. The IC₅₀ of racemate(0.4 nM) and S-enantiomer (<0.1 nM) were more than ten times as potentas the R-enantiomer (13 nM) (Table 8).

TABLE 8 Summary of the Effect of Test Compounds on Growth Factor-inducedHuman Umbilical Vascular Endothelial Cell Invasion VEGF-inducedbFGF-induced HGF-induced Test invasion invasion invasion Compounds IC₅₀Values (μM) IC₅₀ Values (μM) IC₅₀ Values (μM) Racemate 0.00014 0.000420.59 R-enatiomer <0.0001 0.013 0.45 S-enantiomer <0.0001 <0.0001 0.019

8.8 Example 8: Lupus/Fibrosis Mouse Model Study

In this example, sensitivity(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionewas tested in two lupus-prone mouse strains: MRL/MpJ-Faslpr/J mousemodel of systemic lupus erythematosus and NZBWF1/J mouse model ofsystemic lupus erythematosus. The test compound was administered in bothmodels at 30 mg/kg.

In MRL/MpJ-Faslpr/J mouse model of systemic lupus erythematosus,peripheral blood B cells showed no change at week 4. Splenic B cellsshowed 37% increase and anti-double stranded DNA autoantibody levelshowed 25% decrease at week 4.

In NZBWF1/J mouse model of systemic lupus erythematosus, peripheralblood B cells showed 25% decrease at week 4, splenic B cells showed nochange, and anti-double stranded DNA autoantibody level showed 86%increase at week 4.

8.9 Example 9: Dermal Fibrosis Mouse Model Study

In this example,(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionewas tested in the bleomycin-induced skin fibrosis model using bothprophylactic and therapeutic dosing regimens. Bleomycin is an outmodedanticancer therapeutic that has been demonstrated to cause fibrosis inthe lung. In animal models it will similarly induce injury and fibrosisat the site of delivery.

The following abbreviations are used in this example:

Abbreviation or Specialist Term Explanation or Definition ANOVA Analysisof Variance α-SMA Alpha Smooth Muscle Actin CMC Carboxymethyl CelluloseECM Extracellular Matrix NaCl Sodium Chloride PO orally QD Once dailydosing SSc Systemic Sclerosis

DBA/2 mice were used in this study. Eight animals were used pertreatment group in the study. Mice were kept in the animal house understandard conditions with food and water ad libidum.

The vehicle, 0.5% carboxymethyl cellulose (CMC)/0.25% Tween 80, wasprepared in distilled H₂O and dissolved overnight on a magnetic stirrer(add 0.5 g CMC; Sigma #C9481) and 0.25 ml Tween 80 (Sigma #P8074) to99.75 ml to make a total of 100 ml 0.5% CMC/0.25% Tween 80).

The test compound powder was weighed out and suspended fresh daily inthe vehicle 0.5% CMC/0.25% Tween 80, to avoid drug hydrolysis in theaqueous medium. The compound was suspended, not dissolved, in thisvehicle. The formulation was homogenized with a Teflon pestle and mortar(Potter-Elvehjem tissue grinder) using a motorized Eberbach tissuehomogenizer. The daily drug stock concentration used in these studieswas 3 mg/ml.

Bleomycin was obtained from the pharmacy of the University ofErlangen-Nuremberg and freshly prepared once a week. Skin fibrosis wasinduced in 6-week-old DBA mice by local intracutaneous injections of 100μl of bleomycin dissolved in 0.9% NaCl, at a concentration of 0.5 mg/ml,every other day in defined areas of 1.5 cm² on the upper back.

8.9.1 Study Design

The mouse model of bleomycin induced dermal fibrosis is widely used toevaluate anti-fibrotic therapeutics. In this model, a localized dermalfibrosis is induced by intradermal injections with bleomycin every otherday for 3 weeks. This model resembles early, inflammatory stages of SSc.To evaluate potential effects on prevention of fibrosis, treatment wasinitiated simultaneously with the first bleomycin injection. To studythe effect of test compound on prevention of bleomycin-induced dermalfibrosis in vivo, the treatments were divided into following groups:

-   -   Control group: Intradermal injection of NaCl for 3 weeks.        Treatment consisted of administration of the vehicle (0.5%        CMC/0.25% Tween 80).    -   Untreated bleomycin group: Intradermal injection of bleomycin        for three weeks. Administration of the vehicle (0.5% CMC/0.25%        Tween 80).    -   Test compound group: Intradermal injection of bleomycin for        three weeks. The test compound was administered at 30 mg/kg; PO,        QD.    -   Positive control group: Intradermal injection of bleomycin for        three weeks. Injection of Imatinib (50 mg/kg; IP, QD). Imatinib        mesylate has previously been shown to exert potent anti-fibrotic        effects in bleomycin induced dermal fibrosis. See Akhmetshina A.        et al., Arthritis Rheum 2009; 60(1):219-224.

To evaluate regression of fibrosis, a modified model of bleomycininduced dermal fibrosis was used. Mice were pre-challenged withbleomycin to induce a robust skin fibrosis. One group received treatmentwith the test compound, while challenge with bleomycin was ongoing foradditional three weeks. The outcome of this group was compared to micechallenged with bleomycin for six weeks (prevention of furtherprogression) and to mice challenged with bleomycin for three weeksfollowed by NaCl for additional three weeks (induction of regression).The following groups were used in the regression study:

-   -   Control group: Intradermal injection of NaCl for six weeks.        Control treatment consisted of administration of the vehicle.    -   Untreated bleomycin group 1 (regression): Intradermal injection        of bleomycin for three weeks followed by intradermal injections        of NaCl for another three weeks. Treatment consisted of        administration of the vehicle. Untreated bleomycin group 2        (prevention of progression): Intradermal injection of bleomycin        for six weeks. Treatment consisted of administration of the        vehicle.    -   Test compound group: Intradermal injection of bleomycin for six        weeks. The test compound was administered at 30 mg/kg; PO, QD.    -   Positive control group: Intradermal injection of bleomycin for        six weeks.

Injection of Imatinib (50 mg/kg; IP, QD)

8.9.2 Experimental Procedure

Dermal thickness was determined by staining with hematoxylin and eosinand activated fibroblasts by using immunohistochemistry for alpha smoothmuscle actin (α-SMA). The dermal thickness, as determined by themodified Rodnan Skin Score, is currently the most common primary outcomein human clinical trials for anti-fibrotic agents in SSc. Skin sectionswere stained with hematoxylin/eosin for better visualization of thetissue structure. Dermal thickness was analyzed with a Nikon Eclipse 80imicroscope (Nikon, Badhoevedorp, The Netherlands) by measuring themaximal distance between the epidermal-dermal junction and thedermal-subcutaneous fat junction at 4 different skin sections in eachmouse. The evaluation was performed by 2 independent examiners.

For quantification of myofibroblasts, skin sections were deparaffinizedand incubated with 5% bovine serum albumin for 60 minutes. Cellspositive for α-SMA were detected by incubation with monoclonalanti-α-SMA antibodies (clone 1A4; Sigma-Aldrich, Steinheim, Germany) for2 hours at room temperature followed by incubation with 3% hydrogenperoxide for 10 minutes. Goat anti-rabbit antibodies labeled withhorseradish peroxidase (Dako, Hamburg, Germany) were used as secondaryantibodies. The expression of α-SMA was visualized with3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Monoclonalmouse IgG antibodies (Calbiochem, San Diego, Calif.) were used ascontrols.

In addition, the amount of collagen in lesional skin will be measuredwith the SirCol collagen assay; RNA and plasma of all mice were savedfor further analyses.

The test compound significantly decreases dermal thickness of lesionalskin in the bleomycin dermal fibrosis mouse model. The test compound at30 mg/kg; PO, QD significantly prevented dermal thickening byapproximately 25±0.49% (p<0.001, FIG. 42).

Representative photomicrographs of hematoxylin and eosin stained skinsections are shown in FIG. 43. Dermal thickness was assessed bymeasuring the maximal distance between the epidermal-dermal junction andthe dermal-subcutaneous fat junction. The line drawn between thejunction points shows the relative thickness in the treatment groups.

To determine the effect of the treatments on fibroblast activation,α-SMA+myofibroblasts were counted in lesional skin sections. As shown inFIG. 44, the test compound at 30 mg/kg; PO, QD reduced the number ofmyofibroblasts by 24±0.09% (p<0.05). Imatinib at 50 mg/kg reduced dermalthickening by 60±0.34% (p<0.0001) and myofibroblast numbers by 81±0.11%(p<0.0001).

8.9.3 Effect on the Regression of Bleomycin Induced Dermal Fibrosis

The inhibitory effects of the test compound on progression of fibrosiswere also confirmed in the modified bleomycin model designed toinvestigate potential regression of fibrosis. As shown in FIG. 45, thetest compound at 30 mg/kg; PO, QD had no effect on dermal thickening inthe regression model. FIG. 46 shows photomicrographs of representativehematoxylin and eosin stained skin sections. Dermal thickness wasassessed by measuring the maximal distance between the epidermal-dermaljunction and the dermal-subcutaneous fat junction. The line drawnbetween the junction points shows relative thickness in the treatmentgroups. FIG. 47 shows that the test compound did not have an effect onthe numbers of myofibroblasts. Imatinib at 50 mg/kg reduced bleomycininduced dermal thickness by 50±0.3% (p<0.0001) and myofibroblast numbersby 78±0.15% (p<0.0001).

8.10 Example 10: Effect in Tsk-1 Mouse Model

The antifibrotic effects of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dionewere tested in the murine tight skin-1 (Tsk-1) mouse model.

First, the inhibitory effects of the compound on fibrosis in the Tsk-1tight skin mouse model were also investigated. As shown in FIG. 48, thecompound at 30 mg/kg reduced the hypodermal thickening by 45% (p≤0.001),compared to 58% reduction by imatinib at 50 mg/kg (p≤0.001). Theelevated collagen content of the skin, as measured by hydroxyprolinelevels, was reduced 38% by the compound, and 67% by imatinib (p≤0.001).

Next, to examine the effect of the compound on the overexpression ofTGF-β pathway genes in Tsk-1 mouse skin, qRT-PCR was used to measuremRNA levels for six genes: CTGF, PAI-1, COL1A1, α-smooth muscle actin(ALPHA SMA), cartilage oligomeric protein 1 (COMP), and TGFB1.Overexpression was defined as the excess mRNA levels observed in Tsk-1mouse skin compared to normal pa/pa mouse skin. Among these six genes,only four (CTGF, PAI-1, COL1A1, and TGFB1) were expressed to asignificantly higher level in Tsk-1 compared to normal pa/pa mice. Thecompound (30 mg/kg) significantly reduced the overexpression of CTGF andCOL1A1 by 79% (p≤0.001) and 129% (p≤0.001), respectively (FIG. 49).PAI-1 and TGFB1 gene overexpression was not inhibited by the compound.These results indicate that the compound may reduce skin fibrosis byblocking the overexpression of some profibrotic genes within the TGF-βpathway.

To further examine the effect of the compound on the expression offibrotic genes of normal (NL) and systemic sclerosis (SSc) cells, thefollowing procedures were followed:

Cell cultures: Human normal and scleroderma skin fibroblasts werecultured in DMEM containing 10% fetal bovine serum (FBS). The cells weremaintained in a humidified 5% CO2, 95% air incubator at 37° C. Afterconfluence was reached, the cells were harvested with 0.05% trypsin andsubcultured. Cells were used in the 3^(rd)-5^(th) passage.

Experimental design: Cells were grown to 70-80% confluence and placedinto quiescent state by a reduction in serum concentration to 0.4% forovernight. The effects of the compound on TGFβ fibrotic effects and onNL and SSc fibrotic gene expression were tested.

Effects on TGFβ induction of fibrotic gene expression: NL fibroblastswere pretreated with the compound for 30 minutes followed by theaddition of TGF-β1 to observe the effects of the compound on TGFβinduced fibrosis gene expression.

Effects of the compound on SSc-FB: SSc fibrobasts were treated with thecompound at different concentration and gene expression levels ofCollagen 1A1 (COL1), α-smooth muscle actin (α-SMA), fibronectin (FN),matrix metallopeptidase 1 (MMP1), plasminogen activator inhibitor (PAI),and DNA (cytosine-5-)-methyltransferase 1 (Dnmt1) were determined 24hours later by real time PCR.

Quantitative Real-Time PCR: Total RNA was extracted from cells usingRNeasy kit. RNA concentration was measured and 1 ug of RNA was reversetranscribed to cDNA using RT-First strand kit. The cDNA was thenamplified using respective primers and Power SYBR Green PCR Master Mix.The amplicon (150-200 bp) was detected by ABI 7500 Real Time PCR System.All the target genes were normalized to GAPDH and relative fold changeswere calculated. Each sample was assessed in triplicate.

As shown in FIG. 50, the compound dose dependently reduced theexpression of COL1, aSMA and FN mRNAs in SSc fibroblasts. Similarly, theexpression of PAI (FIG. 51) and Dnmt1 (FIG. 52) was also dosedependently reduced by the compound in SSc fibroblasts. As also shown inFIG. 51, the expression of MMP-1 was dose dependently increased by thecompound in SSc fibroblasts. The results demonstrate that the compoundpotently regulates key fibrotic factors, indicating the efficacy of thecompound in the treatment of SSc.

In addition, the levels of cereblon in normal and SSc fibroblasts andskin tissues were examined. As shown in FIGS. 53 and 54, higher levelsof cereblon were observed in both SSc fibroblasts and skin tissues ascompared to the normal tissues. This indicates that an elevated level ofcereblon is involved in SSc.

8.11 Example 11: Targeting Cereblon for B Cell Dyscrasias

The effect of(S)-3-[4-(4-morphlin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydro-isoindo-2-yl]piperidine-2,6-dione(“Compound IA”) on CRBN binding, ubiquitination, and cell proliferationwas profiled. CRBN is a component of the E3 ubiquitin ligase complexincluding CUL4A, DDB1, and ROC-1 and was found to be the molecularbinding target of thalidomide, lenalidomide, and pomalidomide.

Binding studies to CRBN were conducted using thalidomideanalog-conjugated beads in a competitive assay. Endogenous CRBN fromhuman U266 multiple myeloma (“MM”) cells was measured by incubating cellextracts with varying concentrations of either Compound IA orpomalidomide as a positive control. Affinity beads coupled to athalidomide acid analog were incubated with the U266 extracts and, afterextensive washing of the beads, the bound proteins were eluted. CRBNbinding to the thalidomide-coupled affinity beads was determined byquantitative CRBN immunoblot determination.

CRBN ubiquitination was measured in HEK293T cells, which weretransfected with an amino-terminal His-biotin-tagged CRBN construct,then preincubated with compounds for one hour followed by treatment withthe MG132 proteasome inhibitor (to arrest degradation of ubiquitinatedproteins). Cells were lysed and processed to measure CRBN ubiquitinationby SDS-PAGE and immunoblot analysis using an anti-ubiquitin antibody.Cell proliferation studies were conducted in enalidomide-sensitive andrefractory multiple myeloma cells. Lenalidomide-resistant or sensitiveH929 MM cell lines were treated with Compound IA for 5 days, and thencell proliferation and viability were assessed by 7-aminoactinomycin D(“7-ADD”) staining. T-cell costimulation was measured in purifiedprimary human T cells stimulated using immobilized anti-CD3 antibody incell culture for 2 days, and cytokine secretion was measured by ELISA.

Immunoglobulin M and G (“IgG and IgM”) production was measured fromnormal donor peripheral blood mononuclear cells by culturing in thepresence of the B cell differentiation factors recombinant human IL-2(20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), His-tagged CD40 Ligand(50 ng/mL), polyHistidine mouse IgG1 antibody (5 μg/mL), and ODN2006-Human TLR9 ligand (10 μg/mL) for 4 days, followed by IL-2, IL-10,IL-15, and IL-6 (50 ng/mL) for an additional 3 days. IgM and IgG weremeasured by ELISA.

In the competitive CRBN binding studies, preincubation with pomalidomideat concentration of 3 uM resulted in approximately 50% less CRBN boundto the affinity beads, while Compound IA at a concentration of 0.1 μMresulted in similar CRBN binding. CRBN ubiquitination studies in thetransfected HEK293T cells resulted in the following potencies: CompoundIA IC₅₀=0.19 μM; lenalidomide IC₅₀=12.9 μM; and pomalidomide IC₅₀=21.6μM. The IC₅₀ values for inhibition of proliferation by Compound IAshifted from 0.01 μM in the parental H929 cell line and 0.04 μM in theDMSO-treated subclone to 0.51-1.58 μM in the lenalidomide resistantsubclones.

A 50% decrease in cell cycle (S-phase) was evident after 24 hours oftreatment of H929 cells with Compound IA. At 48 hours, Compound IAdecreased expression of survivin and retinoblastoma protein (“pRB”) andincreased expression of the cyclin-dependent kinase inhibitor p27.Compound IA co-stimulated IL-2 production by T cells with an EC₅₀ ofapproximately 0.29 nM, compared with 10 nM for pomalidomide. Compound IAinhibited IgM and IgG production with an IC₅₀ of 0.35 and 2.1 nM,respectively, compared to 17 nM and 63 nM for pomalidomide.

The results indicate that Compound IA binds to CRBN with approximately30-fold higher affinity than pomalidomide, and inhibits CRBNubiquitination with approximately 110-fold greater potency thanpomalidomide in this system. Compound IA is approximately 34-fold morepotent than pomalidomide for co-stimulating IL-2 production by T cells,and is 30 to 48-fold more potent than pomalidomide for inhibitingimmunoglobulin production.

8.12 Example 12: Inhibition of B Cell Differentiation to the Plasmablastand Plasma Cell Lineage

The effects of cereblon (“CRBN”) targeting on the differentiation of Bcells to the plasmablast and plasma cell lineages, an in vitro model ofprimary human B cell differentiation was developed.

CD19+ peripheral blood human B cells from normal donors, or totalperipheral blood mononuclear cell PBMC for patients with systemic lupuserythematosus (“SLE”), were cultured in the presence of interleukin(“IL”)-2, IL-10, IL15, TLR9 agonist, and CD40 L for 4 days, followed byIL-2, IL-6, IL-10 and IL-15 for another 3 days. Cells were counted,viability assessed, and expression of CD20, CD38, CD44, and CD83 weremeasured by flow cytometry. Plasmablast lineage factors IRF-4, BLIMP-1,XBP-1, and IgJ, and germinal center markers PAX-5 and BCL-6 weremeasured by qRT-PCR. Interacellular protein expression was measured bylaser scanning cytometry. Secreted immunoglobulins IgG and IgM weremeasured by ELISA.

In normal B cell cultures,(S)-3-(4-((4-morphlinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione(“Compound I-S”) reduced the percentage of total viable cells in thesecultures after 4 days to 69.6% (P≤0.05) or 35.8% of control (P≤0.001) at20 nM or 200 nM Compound I-S, respectively. Compound I-S decreased thepercentage of viable CD20-CD38+ plasmablasts on day 7 from 30.4% incontrol cultures in a dose-dependent manner to 27.3%, 2.1%, and 0.4% at2 nM, 20 nM, and 200 nM, respectively. On day 7, qRT-PCR analysis showedthat Compound I-S (20 nM) reduced expression of the plasmablast lineagefactors IRF-4, BLIMP-1, XBP-1, and IgJ gene expression to 20.5%, 14.3%,15.1%, and 31.5% of control, respectively (P≤0.001).

By intracellular flow cytometry, Compound I-S (20 nM) significantlydecreased IRF-4 (P<0.5), BLIMP-1 (P<0.05), and XBP-1 (P<0.05) proteinexpression at day 4, but significantly increased BCL-6 (P<0.05) proteinexpression on day 7. By laser scanning cytometry on day 7, Compound I-S(20 nM) reduced CD38+ cell intracellular protein expression of IRF-4(P≤0.001), and BLIMP-1 (P≤0.001), and increased BCL-6 expression(P≤0.05) (n=3). Compound I-S inhibited secreted IgG production with anIC50=1.8 nM (n=3).

In PBMC from SLE patients, Compound I-S (20 nM) had similar effects asin normal B cells, reducing BLIMP-1, XBP-1, and IgJ gene expression to52.8%, 49.2%, and 13.6% of control, respectively (P≤0.001) (n=3).Compound I-S (20 nM) significantly reduced CD38+ plasmablastintracellular protein expression of BLIMP-1 (P≤0.01) and IRF-4(P≤0.001), and increased BCL-6 (P≤0.05) (n=3). Compound I-S inhibitedsecreted IgM and IgG production by SLE patient PBMC with IC50s of 0.9 nMand 3.2 nM, respectively (n=3).

These results demonstrate that targeting of the E3 ubiquitin ligasecomplex substrate co-receptor CRBN with the small moleculeimmunomodulatory compound Compound I-S results in potent inhibition of Bcell differentiation to the plasmablast lineage, as shown by a reductionin the percentage of viable CD38+ cells, a decrease in BLIMP-1, XBP-1,IRF-4, and IgJ gene and protein expression, and inhibition of secretedimmunoglobulin production. These data implicate the CUL4-CRBN complex inthe differentiation of B cells to the plasma cell lineage.

8.13 Example 13: Clinical Studies: Chronic Cutaneous

Sarcoid

Study Design

A clinical study is conducted to examine the efficacy and safety ofmultiple dose levels of Compound I-S in subjects with chronic cutaneoussarcoid. The study employs a randomized, double-blind,placebo-controlled, sequential, dose-ascending design. The study isconducted in patients with CCS and who are receiving chronic stabletherapy for their disease. Subjects may or may not have pulmonaryinvolvement.

Two dose cohorts of Compound I-S(Cohort 1: 0.3 mg PO QD or matchingplacebo and Cohort 2: 0.6 mg PO QD or matching placebo) are evaluatedusing a sequential, dose-ascending design. Thirty-two subjects arerandomized into each dose cohort at a 3:1 ratio (active to matchingplacebo). Subjects remain on their assigned treatment for up to 12weeks. Enrollment are terminated when approximately 16 subjects havebeen randomized into each cohort.

The first group (Cohort 1) of approximately 16 subjects who meet thestudy entry criteria are randomized to 0.3 mg PO QD dose or matchingplacebo in a 3:1 ratio. After approximately 13 subjects in this cohorteither complete the 4 weeks of treatment or discontinue the study early,a review of all blinded safety data is performed. If the safety profileis determined acceptable for Cohort 1, the enrollment of the remainingsubjects into Cohort 1 continues and the randomization of 16 subjectsinto Cohort 2 (0.6 mg PO QD dose or placebo) in a 3:1 ratio commences.Subjects have regularly scheduled visits (weekly for the first month,then every 2 weeks for the next 2 months) to assess activity, safety,tolerability, efficacy, PK and PD of Compound I-S.

Study Population

Eligible subjects must have been diagnosed with CCS with or withoutpulmonary involvement. Subjects also have the following: i) documentedhistological diagnosis of CCS of ≥12 months duration; ii) CS biopsydemonstrating a confirmed diagnosis at screening; iii) minimum ModifiedSarcoidosis Activity and Severity Index (MSASI) of ≥1 induration ofobserved lesion at randomization; iv) ≥2 lesion(s) >1 cm in longestdimension; v) no improvement in CCS ≥3 months prior to randomization (asdeemed by the investigator), despite chronic therapy; v) forced vitalcapacity (FVC) of ≥45% of predicted normal value; and/or vi) estimatedGlomerular Filtration Rate (eGFR)=≥60 mL/min. (calculated by MDRD[Modification of Diet in Renal Disease]).

Length of Study

This study consists of 3 phases of a maximum duration of 25 weeks:screening phase (up to 5 weeks), treatment phase (12 weeks), andpost-treatment observational follow-up phase (8 weeks).

Efficacy Assessments

Efficacy assessments in this study include the following:

Modified Sarcoidosis Activity and Severity Index (MSASI): Subject'sindex lesion is evaluated and scored during screening using the ModifiedSarcoidosis Activity and Severity Index (MSASI). The symptom domains oferythema, induration and desquamation are scored as follows: 0=None;1=Slight; 2=Moderate; 3=Severe; and 4=Very Severe. The score forerythema and/or induration of the index lesion must be ≥1 for studyeligibility.

The area score of the index lesion is assessed as follows: 0=0%; 1=1-9%;2=10-29%; 3=30-49%; 4=50-69%; 5=70-89%; and 6=90-100%.

The MSASI is assessed at screening, baseline, Weeks 4, 8 and 12.

Physician's and Subject's Global Assessment of Disease Activity: ThePhysician's (Evaluator's) and Subject's Global Assessments of DiseaseActivity are recorded as a single vertical mark on a 100-mm visualanalog scale (VAS), and serve as the subject and physician global VASassessments of disease severity. The evaluation measures the degree ofdisease severity at the time of the examination using a 0-100 mm VASscale. The subject global assessment of disease severity VAS iscompleted prior to the completion of investigator assessments. Thephysician global assessment of disease severity VAS is performed by thesame trained investigator or sub-investigator at each visit. Thephysician's and the subject's global assessment of disease activity willbe assessed at baseline, Weeks 1, 2, 4, 8, and 12.

6-Minute Walking Test: The 6MWT has emerged as an important addition tothe prognostic evaluation of sarcoid. Published data suggest that thechanges in 6MWT are clinically meaningful measures of a patients' dailyactivity, and is highly relevant to their quality of life (Lama et al.,“Prognostic value of desaturation during a six minute-walk test inidiopathic interstitial pneumonia,” Am. J. Respir. Crit. Care. Med.2003, 168:1084e90; Hallstrand et al., “The timed walk test as a measureof severity and survival in idiopathic pulmonary fibrosis,” Eur. Respir.J. 2005, 25:96e103). The 6MWT is assessed at baseline, and at Weeks 4, 8and 12. The Borg Scale can be used in conjunction with the 6 MWT asfollows: 0=no breathlessness at all; 0.5=very very slight (justnoticeable); 1=very slight; 2=slight breathlessness; 3=moderate;4=somewhat severe; 5=severe breathlessness; 7=very severebreathlessness; 9=very very severe (almost maximum); and 10=maximum.

Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F):Chronic fatigue is often an accompanying component of CCS. The FACIT-Fquestionnaire is designed to measure anemia-related fatigue in cancerpatients and is also gaining popularity in clinical trials outside ofthe cancer setting. The FACIT-F scale was used to demonstratesignificant improvement in sarcoidosis-associated fatigue (Lower et al.,“Double-blind, randomized trial of dexmethylphenidate hydrochloride forthe treatment of sarcoidosis-associated fatigue,” Chest 2008,133(5):1189-95).

The questions fall into five subsets: physical well-being (PWB),social/family well-being (SWB), emotional well-being (EWB), functionalwell-being (FWB) and additional concerns (fatigue subset). Each of thesesubsets consists of questions with five responses ranging from zero tofour, depicting “not at all” to “very much”. The FACIT-F is completed atbaseline, Weeks 4, 8, and 12.

Dermoscopy for Lesion Assessment: This is a noninvasive method thatallows for an in vivo evaluation of the observed lesion. This techniquecompares the baseline lesion versus Week 12. The same lesion should beused for this assessment.

Physician's Overall Skin Response Assessment: The index lesion is asingle, defined area of involvement that exhibits the most severe CS atscreening and is located in such as position as to facilitate visualevaluation and measurement. The overall lesion evaluation for responderstatus is assessed by the investigator as follows: 1=markedly worsened;2=worsened; 3=unchanged; 4=near resolution; and 5=complete resolution.The measurement of index lesion includes measurement of thecross-sectional diameter of the index lesion (the diameter at the widestpoint of the lesion) and measurement of the diameter perpendicular tothe first measurement.

Photography and Physician's Photographic Assessment of the Index Lesion:Photographic assessments are conducted in a subset of subjects atdesignated sites to provide supportive evidence of efficacy.Investigators will identify an area of the body where the index lesionis located, and take photographs of that same index lesion at baseline,Week 8 and Week 12. The investigator reassesses the same index lesionphotographed at baseline, at both the Week 8 and Week 12 photographs.The investigator indicates his/her response, for example, as follows:much better after therapy, somewhat better after therapy, unchanged,somewhat worse after therapy, or much worse after therapy.

Pharmacokinetics: subjects participate in PK samplings.

Skin biopsy of Non-facial Lesion: All subjects have a skin biopsyperformed on a CS lesion at the screening visit to reconfirm that theyhave active cutaneous sarcoid at study entry. This skin biopsy taken atthe screening visit also serves as the baseline biopsy for thosesubjects who agree to having the optional biopsy collected at Week 12.

Two 4 mm diameter skin biopsies of the non-facial lesion are obtained.Evaluations includes changes in granuloma size and number, and epidermalthickness, and IHC staining for cells such as Giant cells, T cellstaining (CD4 and CD8), and Dendritic cells/Langerhans staining (CD1a,CD83, DC-LAMP).

Blood Biomarker Analyses: The blood biomarker sampling is collected atthe same time as the routine clinical laboratory blood draws, prior tothe subject's morning dose. Blood samples are drawn at baseline, Weeks4, 8, and 12 of the Treatment Phase, and at Weeks 16 and 20 of thePost-Treatment Observational Follow-up Phase. Evaluations include: serumACE, IgG, 25-hydroxy vitamin D (25-OH-vit D), 1,25-dihydroxy vitamin D(1,25-vitD) levels; plasma biomarkers (e.g., IL-12, sIL2R and TNF-α);peripheral blood total lymphocyte counts and B cell subsets and T cellactivation markers (such as but not limited to HLA-DR, CD45RA, CD45RO,CD69, IL-2R, CD80, CD86) via flow cytometry.

Pulmonary Function Tests: FVC and FEV₁ are measured at screening,baseline, Weeks 8 and 12 of the Treatment Phase. FVC measures the totalamount of air that can be forcibly blown out after full inspiration. FVCis measured in liters. FEV₁ measures the amount of air that can beforcibly blown out in one second. FEV₁ is measured in liters. Along withFVC, FEV₁ is considered one of the primary indictors of lung function.

Chest Likert: The radiologist compares two radiographic films from thesame patient. The films are taken at screening (this film also serves asthe baseline image) and again at Week 12. The radiologist scores the twofilms on a Likert scale of 1 to 5 (markedly worsened, worsened,unchanged, improved, and markedly improved).

St. George's Respiratory Questionnaire (SGRQ): The SGRQ is astandardized self-completed questionnaire for measuring impaired healthand perceived well-being (‘quality-of-life’) in airways disease. It hasbeen designed to allow comparative measurements of health betweenpatient populations and quantify changes in health following therapy.The SGRQ is completed only for those subjects who have pulmonaryinvolvement at baseline, Weeks 4, 8, and 12 of the Treatment Phase.

Baseline and Transition Dyspnea Indices (BDI/TDI): The BDI/TDI provideinterview-based measurements of breathlessness related to activities ofdaily living. The BDI is a discriminative instrument that includesspecific criteria for each of three components (functional impairment,magnitude of task and magnitude of effort) at a single point in time.The TDI is an evaluative instrument that includes specific criteria foreach of three components to measure changes from a baseline state. Atbaseline, dyspnea is rated using five grades from 0 (severe) to 4(unimpaired) for each category to form a baseline score (0 to 12).During the transition period, the TDI assesses changes in dyspnea usingseven grades ranging from −3 (major deterioration) to +3 (majorimprovement). The ratings for each of the three categories are thenadded to form a total transition focal score (range, −9 to +9). The BDIis performed at baseline, and the TDI is performed at Weeks 4, 8, and 12of the Treatment Phase.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1. A method for treating or managing an immune-related disease or an inflammatory disease comprising: a) identifying a patient suffering from the disease as having overproduction of IgG; and b) administering to the patient an effective amount of a compound, wherein the compound is (S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione of the formula:

or a pharmaceutically acceptable salt thereof, wherein the disease is lupus pernio or sarcoidosis.
 2. (canceled)
 3. The method of claim 1, wherein the compound is (S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione.
 4. The method of claim 1, wherein the compound is (S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride.
 5. The method of claim 1, wherein the disease is chronic cutaneous sarcoidosis.
 6. The method of claim 5, wherein the chronic cutaneous sarcoidosis is with pulmonary involvement.
 7. The method of claim 5, wherein the chronic cutaneous sarcoidosis is without pulmonary involvement.
 8. The method of claim 1 further comprising administering a second active agent, which is an anti-inflammatory or immunomodulatory compound.
 9. The method of claim 1, wherein the effective amount is about 0.005 mg/kg to about 10 mg/kg of the patient's body weight.
 10. A method for reducing or inhibiting a symptom of sarcoidosis comprising: a) identifying a patient suffering from sarcoidosis as having overproduction of IgG and b) administering to the patient an effective amount of a compound, wherein the symptom is selected from the group consisting of (i) granulomas formulation, (ii) lupus pernio, (iii) skin lesions, (iv) dyspnea, (v) dry cough, (vi) chest pain, and (vii) fatigue, and wherein the compound is (S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione of the formula:

or a pharmaceutically acceptable salt thereof. 11-15. (canceled)
 16. A method for improving the Modified Sarcoidosis Activity and Severity Index (MSASI) score, the Physician's and Subject's Global Assessment of Disease Activity score, the 6WMT score, the FACIT-F score, the Dermoscopy for Lesion Assessment score, the Physician's Overall Skin Response Assessment score, the lesion, one or more biomarkers associated with sarcoidosis, forced vital capacity (FVC), the amount of air that can be forcibly blown out in one second (FEV₁), the chest radiographic Likert score, the Saint George's Respiratory Questionnaire score, or the dyspnea index (BDI)/transition dyspnea index (TDI) of a patient having sarcoidosis, comprising: a) identifying the patient as having overproduction of IgG and b) administering to the patient an effective amount of a compound, wherein the compound is (S)-3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione of the formula:

or a pharmaceutically acceptable salt thereof. 17-21. (canceled)
 22. The method of claim 1, wherein the compound is administered at a dosage in the range of from about 0.3 mg once per day to about 0.6 mg once per day.
 23. The method of claim 22, wherein the dosage is about 0.3 mg once per day.
 24. The method of claim 22, wherein the dosage is about 0.6 mg once per day.
 25. The method of claim 1, wherein the administration of the compound continues for a period of about 12 weeks.
 26. An intermediate of the structure: 